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Intact Protein Sequencing Using ETD and PTR in a Dual-Pressure

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Intact Protein Sequencing Using ETD and PTR in a Dual-Pressure Powered By Docstoc
					Intact Protein Sequencing Using ETD and PTR in a Dual-Pressure Linear Ion Trap
Zhiqi Hao, Jae C Schwartz and Andreas FR Hühmer
Thermo Fisher Scientific, San Jose, CA, USA

 Overview                                                                                                                                              Results                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    FIGURE 2. Sequencing of intact ubiquitin using ETD-PTR.                                                                                                                                                      FIGURE 3. Top-down sequencing of myoglobin (top) or carbonic anhydrase
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               (bottom) using LTQ Velos ETD with PTR. Results were from 10 min data averaging.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          FIGURE 4. Improved identification from top–down sequencing using ETD with
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          PTR in a dual-pressure linear ion trap.
 Purpose: To evaluate the performance of proton transfer reaction (PTR) in a linear ion
 trap for charge reduction following electron transfer dissociation (ETD) and to investigate
 the utility of ETD with PTR for intact protein sequencing.                                                                                             FIGURE 1. Electron transfer and proton transfer for charge reduction
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        LTQ Velos ETD spectrum of intact myoglobin (16.9KD)
 Methods: Experiments were performed using LTQ XL ETD ion trap and LTQ Velos dual-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                LTQ Velos ETD PTR spectrum of intact ubiquitin                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Total Number of c / z Ions Identified *
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               9 0


 pressure linear ion trap mass spectrometers, both equipped with ETD and PTR, under                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            8 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               8 0




 LTQ 2.6 instrument control software with developer’s kit. Benzoic acid anions, which are                                                                Charge reduction using electron transfer or proton transfer
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               7 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               7 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               6 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                ETD 5 ms PTR 25 ms                                                                                                                                                                                                                                    1 4 1 0 .0 2

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1 5 8 5 .2 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1 6 7 1 .3 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1 8 7 9 .8 8


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1 9 9 4 .4 6                          250




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Relative Abundance
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               6 0                                                                                                                                                                8 0 4 .8 2




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Relative Abu
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1 2 0 6 .8 2

 generated in the chemical ionization source at the rear of the instrument, were used as                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       5 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               5 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 1 4 6 7 .7 4                                    1 7 3 2 .3 8


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   * The same product ions of different charge states were counted as different ions.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             50                                                                                                                                                                                                                4 5                                                                                                                                                                                                                           1 1 3 5 .7 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       1 2 3 0 .8 4                                                      1 5 3 4 .1 6                                     1 7 6 0 .4 2



 PTR reagent.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1 8 5 1 .3 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               4 0                                                                                                                                                                                                                                                                                 1 3 2 3 .9 2

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               3 5                                                                                                                                                       7 6 2 .4 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       9 0 0 .5 4           1 0 4 2 .6 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                200
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    •                              Charge reduction can be
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               3 0




 Results: When intact protein ETD analysis is performed on a unit-resolution instrument,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         277.14                ETD 10 ms PTR 25 ms                                                                                                                                                     2 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               2 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              4 4 7 .1 6                                6 5 4 .8 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              9 1 9 .5 6




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Num ber of Ions
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   achieved by electron transfer                                                                                                                             40                                                                                                                       1428.90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               1 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               1 0                                                 1 8 8 .0 8      2 7 5 .0 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          6 1 5 .7 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   7 1 2 .3 8




 the resulting spectra are information rich, yet contain multiply charged product ions which
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3 9 0 .1 4                    5 6 2 .7 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   5


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   or by proton transfer.                                                                                                                                                                                                                                                                                                                                                                          0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2 0 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2 9 8 .0 8

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        4 0 0                                   6 0 0                                  8 0 0                            1 0 0 0                                               1 2 0 0                                                  1 4 0 0                             1 6 0 0                              1 8 0 0                         2 0 0 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                150
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  m   /z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    LTQ XL ETD PTR
 can not be resolved adequately. PTR following ETD reduces charge carried by product                                                                         Benzoic Acid                                                                                                                                                                                                                                     Fluoranthene
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    •                              The end product of the proton                                                                                                                             30                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     LTQ Velos ETD PTR
 ions so that they are resolved at unit resolution. Using different PTR time, ETD-PTR of
                                                                                                                                                                                                                      O
                                                                                                                                                                                                                                                                                [M + 4H]4+ + A-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   transfer pathway has the                                                                                                                                                                                                                                                                                                                                                 100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 c41+4
 intact proteins generates very informative and well-resolved spectra. The improved                                                                                                                                           O
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  c40+4                                                                                                                                                                                                                  100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   expected mass while that of                                                                                                                               20                           390.24                                                                                                      1663.22
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        c31+3                                                                                                                                             c59       +5
 sensitivity, resolution, and faster scan rate of the LTQ Velos mass spectrometer resulted                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             960.26                     1347.64                                                                                                                                                                                                                                    c59+6                                                                                                                                                        90

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   the electron transfer pathway                                                                                                                                                                                                                                   1136.92                                                                                                   80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                50
 in extensive sequence coverage of intact proteins of up to 30KDa. With product ions of                                                                                                                                                                                                                                                                                                                                                                                                                                                            contains extra hydrogen.                                                                                                                                                                                                                                                                                                                                                  70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          80


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        537.32            790.62                                            1274.04           1469.76                    1858.98                                                                                                   c48+5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         c47+4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             10 149.06




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Relative Abundance
 +5, +6 and even +7 charge resolved in full, zoom scan mode, the LTQ Velos instrument                                                                                                                                [M +                        3H]3+                                                                                                                      [M +                     4H]3+•                                                                                                                                                                                                                                                                                                                                                           653.48 898.66 1099.36
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             60
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          z31+3                                                                                                                                                                  z37+3




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Relative Abundance
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          60


 identified many c’/z. product ions as big as 10 kDa in the complex spectrum of intact                                                                                                                                                                                                                                                                                                                                                                                                                              •                              Partitioning between the two                                                                                                                                                                                                                                                                                                                                              50                                                                                                                                                                                                                                                                                                                                                               z60+5                                                                                              0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      ubinquitin                        myoglobin                    carbonic anhydrase
 myoglobin and carbonic anhydrase in a single 10 minute experiment. Number of                                                                                                                                                                                                                                                                                                                                                                                                                                                                      pathway depends on the                                                                                                                                                                                                                                                                                                                                                    40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             z60+6                                                                                                                                                                                        40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       (8.6 KD)                                                          (29.0KDa)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      (16.9KD)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   reagent. The proton transfer                                                                                                                                                                                                                                                                                                                                              30
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          30

 identified ions, thus the sequence coverage was significantly improved in LTQ Velos                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       200                  400            600         800                              1000           1200       1400            1600              1800           2000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   mechanism is the major                                                                                                                                                                                                                                                                                                                                                    20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          20

 compared to LTQ XL.                                                                                                                                                                                                       [M + 2H]2+                                                                                                                                               [M + 4H]2+••                                                                                                                                                   pathway for benzoic acid and                                                                                                                                                                                                                                m/z                                                                                                           10                                                                                                                                                                                                                                                                                                           10


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  0                                                                                                                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   electron transfer is the major                                                                                                                                                                                                                                                                                                                                                                  1090                                    1095                 1100                  1105                 1110                      1115                      1120                 1125               1130                                        1135                                                 1320                       1325                          1330                      1335                      13



 Introduction
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   m /z                                                                                                                                                                                                                                  m/z
                                                                                                                                                        Proton Transfer Pathway                                                                                                                                                                          Electron Transfer Pathway                                                                                                                                                                 pathway mechanism for
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           G L S D G E W Q Q V L N V W G K V E A D I A G H G Q E V L I R
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   fluoranthene.
 Electron transfer dissociation (ETD) is an beneficial tool for intact protein analysis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      •                  Intact proteins of 9-29 kDa are used to evaluate the performance of ETD with PTR
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       MH+7                                                                                                         z74+5                                                                                                  L F T G H P E T L E K F D K F K H L K T E A E M K A S E D L K
 because it is relatively insensible to the size, amino acid composition and post-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   90                                                                                           90                                                                            z59+4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           in a unit resolution instrument for top-down sequencing.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     80                                                                                           80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           K H G T V V L T A L G G I L K K K G H H E A E L K P L A Q S H
 translational modifications of proteins, therefore randomly cleaves protein / peptide                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       •                  For the LTQ XL ETD ion trap with PTR, extensive sequence coverage is obtained
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     70                                                                                           70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           A T K H K I P I K Y L E F I S D A I I H V L H S K H P G N F G
 backbone bonds. ETD of intact proteins performs with high efficiency, generating very                                                                       ETD or ETD-PTR spectrum of histone 3 peptide (M+6H)6+                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              for ubiquitin and myoglobin proteins while less ions are indentified in a larger protein




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Relative Abundance
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     60                                                                                           60
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           z73+5 / C72+5                                                                                                                                                   A D A Q G A M T K A L E L F R N D I A A K Y K E L G F Q G                                                                                                                                                                                                                                                                                                                                                            such as carbonic anhydrase due to the limited resolving power.
 informative, yet extremely complex spectra which contain highly charged product ions                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                50                                                                                           50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             c44   +3
 that are difficult, or even impossible to resolve at unit resolution. Proton transfer reaction                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 For the LTQ Velos dual-pressure ion trap the number of identified c’/z. ions is 2-3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     40                                                                                           40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             •
 (PTR) following ETD was developed to reduce spectral complexity. PTR removes                                                                                                                              ARTKQTARKsTGGKAPRKQLAGGK-BIOTIN                                                                                                                                                                                                                                                                                                                        MH+ = 2802.4                                                                                                                       30
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           MH-H2O+7/
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  30
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              +5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     +4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         times higher due to improved sensitivity, resolution, as well as faster scan rate.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  20
 protons from the multiply charged product ions, generating a simplified spectrum that
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           MH-NH3+7
                                                                                                                                                                                                                     1 4 2 1 .8 6                                                                                                                                                     1 4 7 8 .9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     10
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 c75+7                                                                            10                                                                                                                                                                               LTQ Velos ETD spectrum of intact carbonic anhydrase (29KD)                                                                                                                                                                                                                                                                                                                                •                  The new LTQ Velos instrument provides significantly improved sequence coverage
 contains product ions of resolved charge states at unit resolution. PTR has recently been
                                                                                                                                                                                    1 0 0

                                                                                                                                                                                        9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2 7 9 0 .6 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1 00
                                                                                                                                                                                        8 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   0
                                                                                                                                                                                        7 0                                                                            1 4 4 0 .8 0                                                                                                                                                                                   9 0                                                                                                                                                                                                                             0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1635          1640        1645          1650          1655           1660           1665                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    for all proteins investigate with a particularly large benefit for larger proteins.
                                                                                                                                                               Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 1216     1218     1220         1222    1224   1226
 implemented, under software control, in the LTQ XL ion trap and the new LTQ Velos
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      8 0                                              2 7 8 9 .6 4
                                                                                                                                                                                        6 0                                    1 4 2 3 .9 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      7 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          m/z                                                                                                 m /z




                                                                                                                                                                                                                                                                                                                                                                                                                                                Relative Abundance
                                                                                                                                                                                        5 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      6 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  8 5
                                                                                                                                                                                                                                    1 4 2 5 .8 2
                                                                                                                                                                                        4 0                                                                                                                                                                                                                                                                                                                                                  2 7 9 1 .6 6                                                                 2 8 0 6 .6 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      5 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2 7 9 3 .5 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           8 0


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   ETD 5 ms PTR 40 ms
                                                                                                                                                                                        3 0



 dual-pressure linear ion trap. Shown below is the instrument configuration of LTQ Velos
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      4 0                                                                                                                                        2 8 0 5 .4 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   2 8 0 7 .5 8
                                                                                                                                                                                        2 0                                                1 4 3 1 .8 0
                                                                                                                                                                                                                                                             1 4 3 7 .9 8                          1 4 4 8 .8 2                                   1 4 6 4 .8 2
                                                                                                                                                                                                                                                                                                                                                                         1 4 7 3 .8 6                                                                                 3 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           7 5
                                                                                                                                                                                                                                                                                                                    1 4 5 3 .0 4                                                                                      1                                                      2 7 8 0 .6 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2 0                                          2 7 8 8 .5 6                             2 7 9 4 .6 0                                                                 2 8 0 8 .3 0
                                                                                                                                                                                        1 0                                                                                                                                1 4 5 4 .8 0                          1 4 7 1 .9 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2 7 8 7 .7 2                                                     2 7 9 7 .6 4                2 8 0 4 .7 8                                      2 8 1 3 .2 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           7 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2 7 8 3 .7 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1 0                                                                                                                2 8 0 0 .2 8
                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                           1 4 4 0 .8 4
                                                                                                                                                                                    1 0 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  6 5


 ion trap with ETD and PTR. The new technologies in LTQ Velos instrument not only
                                                                                                                                                                                                                                                                                                                                                                                                                                                                        0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2 8 0 3 .3 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1 00




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  Relative Abundance
                                                                                                                                                                                        9 0                                                                                                                                                                                                                                                                                                                                                                                                                      2 8 0 4 .3 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           6 0


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         10 ms ETD on the LTQ VelosTM instrument generates a very informative spectrum of
                                                                                                                                                                                                                     1 4 2 1 .7 8                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   1 9 9 5 .7 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      9 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          8 7 8 .5 0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             •
                                                                                                                                                                                        8 0                                                                                                                                                                                         1 4 7 7 .8 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      8 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  5 5
                                                                                                                                                                                        7 0                                                                                                                                                                                                1 4 7 8 .7 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               2 8 0 2 .3 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1 1 2 6 .1 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      7 0
                                                                                                                                                                                                                                                                                     1 4 4 1 .7 8                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          5 0

 improve the sensitivity by 5-10 times, but also improve trapping, isolation and
                                                                                                                                                                                        6 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      6 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2 8 0 5 .3 6                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1 4 1 0 .2 2
                                                                                                                                                                                                                            1 4 2 2 .9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          7 5 1 .0 2
                                                                                                                                                                                        5 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           4 5                                                                                                                                                                                                          1 2 0 0 .9 8                                                               1 5 0 7 .0 2

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         intact ubiquitin.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      5 0

                                                                                                                                                                                        4 0                                    1 4 2 3 .9 4                                                                                                                                                        1 4 8 0 .8 8                                                       4 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1 6 0 1 .1 4
                                                                                                                                                                                                                                                             1 4 3 7 .8 8                                                                                                                                                                                                                                      2 7 8 7 .2 0       2 7 8 9 .2 4                                                                              2 8 0 6 .2 4                                                                                                                                                                                                                                                                                                                                   4 0                                                                                                     7 1 2 .6 8                                                                                                       1 2 5 9 .4 6
                                                                                                                                                                                        3 0                                                                                                                                                                                                                                                                           3 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1 0 0 4 .5 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           1 9 2 6 .1 8

 dissociation efficicencies. The dual-pressure trap provides two times faster overall scan                                                                                              2 0                                                                                                                             1 4 5 5 .5 4           1 4 6 3 .9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2 7 9 0 .3 0                                                                                 2 8 0 7 .3 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           3 5                                                                                                                                                                  1 0 6 8 .6 2
                                                                                                                                                                                                                                                   1 4 3 4 .4 6                              1 4 4 7 .8 6                                                                1 4 7 4 .8 6
                                                                                                                                                                                                                                                                                                                                                                                                        1 4 8 3 .8 4                                                                                 2 7 8 6 .2 4                         2 7 9 1 .2 6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      7 7 3 .8 8
                                                                                                                                                                                        1 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2 7 7 9 .7 6                                                           2 7 9 3 .1 4
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       2 8 0 8 .2 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           3 0                                                                                                                                                                                                                                                                                                               1 6 3 9 .2 0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Conclusions
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2 7 9 7 .8 8   2 8 0 1 .3 4                                            2 8 1 0 .2 8   2 8 1 3 .0 2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                        0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    5 0 6 .7 0         5 8 8 .3 0
                                                                                                                                                                                                           0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1 7 3 1 .1 6

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             •           A subsequent 25 ms PTR reaction reduces charge states of product ions below +7,
                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2 78 0                  2 7 85                        2 7 9 0                   2 7 9 5                      2 8 0 0                  28 0 5                       2 81 0
                                                                                                                                                                                                                 1 4 2 0                       1 4 3 0                     1 4 4 0                      1 4 5 0                         1 4 6 0                   1 4 7 0                  1 4 8 0                                                                                                                                                                         m /z                                                                                                                                                                                                                                                                                                                                                                                            2 5                                                4 2 1 .1 6                                                                                      9 4 4 .7 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   1 3 3 1 .8 8


 speed compared to LTQ XL instrument. Here, the performance of ETD PTR for intact
                                                                                                                                                                                                                                                                                                                               m   /z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1 8 9 0 .5 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           2 0
                                                                                                                                                                                                                                                                              z 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2 8 4 .0 8
                                                                                                                                                                                                                                                                              4 7 1 .6                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     1 5
                                                                                                                                                                                                           1 0 0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         which are well resolved in LTQ Velos instrument in full scan zoom mode.                                                                                                                                                                           1 0                                        3 7 5 .1 8

 protein analysis is compared between the LTQ XL and LTQ Velos instruments.
                                                                                                                                                                                                               9 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               5          2 0 0 .0 0
                                                                                                                                                                                                               9 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           PTR following ETD reduces charge carried by product ions, thus the complexity of
                                                                                                                                                                                                               8 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2 0 0                         4 0 0                               6 0 0                                 8 0 0                           1 0 0 0                                              1 2 0 0                                              1 4 0 0                              1 6 0 0                           1 8 0 0                          2 0 0 0


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             •           A total of 221 c’ and z. ions were identified with some of them present in the spectrum
                                                                                                                                                                                                               8 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                m /z
                                                                                                                                                                                                               7 5
                                                                                                                                                                                                                                                                                                                                    + 1
                                                                                                                                                                                                                                                                                                                                c


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           the ETD spectrum.
                                                                                                                                                                                                                                                                                                                                    7
                                                                                                                                                                                                               7 0                                                                                                              7 7 4 .0




 Methods                                                                                                                                                                                                       6 5                                                                                                                                    z 7
                                                                                                                                                                                                                                                                                                                                                         + 1

                                                                                                                                                                                                                                                                                                                                                      9 1 1 .9                                                                                                                                                                                                                                                                                                                                                           as multiply charged ions.
                                                                                                                                                                                     Relative Abundance




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         c16+2
                                                                                                                                                                                                               6 0


                                                                                                                                                                                                                                                                                                                                                                                                                                                                        ETD100 ms
                                                                                                                                                                                                               5 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  100

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 c32+3
                                                                                                                                                                                                               5 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    100
                                                                                                                                                                                                               4 5

                                                                                                                                                                                                               4 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           •           The largest c’ and z. ions (c’75 of 8502.6 Da and z.75 of 8413.6 Da) are both identified
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         95

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      95
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Charge reduction of product ions can also be achieved by extending the ETD
 Standard peptides were purchased from Anaspec. Intact proteins were purchased from
                                                                                                                                                                                                                                                             + 1                          c 4
                                                                                                                                                                                                                                                                                             + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      90
                                                                                                                                                                                                                                                           c 2                                                                                             + 1                                 + 1
                                                                                                                                                                                                                                                                                                                                                                                           c 1 0                                                                                                                                                             z 2 0                                                                                                                                                                                                                                                                                                                                                                                       85


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           reaction time. However extended ETD reactions produce c’ /z. ions containing one
                                                                                                                                                                                                               3 5                                         2 4 4 .8                       4 7 3 .9                                                     c 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2 3 3 0 .7                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               85

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         as multiply charged species.
                                                                                                                                                                                                                                                                                                                  + 1                                                                      1 2 2 5 .9
                                                                                                                                                                                                               3 0
                                                                                                                                                                                                               2 5
                                                                                                                                                                                                                                                                  + 1
                                                                                                                                                                                                                                                                c 3
                                                                                                                                                                                                                                                                3 4 5 .8
                                                                                                                                                                                                                                                                                                            c 6
                                                                                                                                                                                                                                                                                                            7 0 2 .9
                                                                                                                                                                                                                                                                                                                                                       9 3 0 .2
                                                                                                                                                                                                                                                                                                                                                                + 2
                                                                                                                                                                                                                                                                                                                                                              z 1 7
                                                                                                                                                                                                                                                                                                                                                              1 0 1 6 .0
                                                                                                                                                                                                                                                                                                                                                                                                                          + 1
                                                                                                                                                                                                                                                                                                                                                                                                                      c 1 1
                                                                                                                                                                                                                                                                                                                                                                                                                      1 3 2 6 .6
                                                                                                                                                                                                                                                                                                                                                                                                                                + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                         + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                       c 1 2
                                                                                                                                                                                                                                                                                                                                                                                                                                       1 3 8 3 .6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    z 1 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2 1 0 1 .7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    c 2 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2 3 3 3 .6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         75
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      80                                                        c43+4
 Sigma. Desalted intact protein was diluted in acetonitrile / water / formic acid (50:50:0.1)                                                                                                                  2 0                                                                                                                                                    z 8
                                                                                                                                                                                                                                                                                                                                                                            + 1
                                                                                                                                                                                                                                                                                                                                                                                                                          c 1 3
                                                                                                                                                                                                                                                                                                                                                                                                                          1 4 4 0 .8                                          + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                            z 1 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    c25+3                                                                                            75


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           or more extra hydrogen, especially for ions at higher m/z. Spectra from extended
                                                                                                                                                                                                                                                                                                                                                                      1 0 6 8 .1                                                                                            1 7 4 6 .3                                                                                                                                                                                                                                                                                                                                                                                                                                                                   70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      70
                                                                                                                                                                                                               1 5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         65
                                                                                                                                                                                                               1 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    65

 to a final concentration of 1 to 5 pmol/μl. The sample was directly infused using static




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Relative Abundance
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         60




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 Relative Abundance
                                                                                                                                                                                                                 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    60

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           ETD reactions also look noisy due to secondary fragmentations.
                                                                                                                                                                                                                                                                                 + 1


                                                                                                                                                                                                               0
                                                                                                                                                                                                           1 0 0
                                                                                                                                                                                                                                                                              z 3
                                                                                                                                                                                                                                                                              4 7 1 .5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   55
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      c33+4                                                                                                                                                           55



 nanospray with a 4 micron tip (PicotipTM, New Objective). ETD with PTR was performed
                                                                                                                                                                                                               9 5
                                                                                                                                                                                                               9 0
                                                                                                                                                                                                               8 5
                                                                                                                                                                                                                       0                                                              5 0 0                                                             1 0 0 0
                                                                                                                                                                                                                                                                                                                                                                                                                  m
                                                                                                                                                                                                                                                                                                                                                                                                                       1 5 0 0
                                                                                                                                                                                                                                                                                                                                                                                                                      /z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       2 0 0 0                                                                                 2 5 0 0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Sequence coverage from a 1 min data acquisition                                                                                                                                                                      50

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         45

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        c34+4                                                                         50
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      45

                                                                                                                                                                                                               8 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   c42+5                                                                                                                                                                                              40

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           By subtracting protons, PTR following ETD generates charge reduced c’/z. ions that
 using Thermo Scientific LTQ XL ETD and LTQ Velos ETD mass spectrometers with PTR                                                                                                                              7 5


                                                                                                                                                                                                                                                                                                                                                                                                                                                                     ETD10 ms / PTR100 ms
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         35

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         30
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           z8+1                                 c43+5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      35

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      30
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               c31+3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       LTQ XL – 10 ms ETD with 100 ms PTR
                                                                                                                                                                                                               7 0



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           do not contain extra hydrogen. These ions show the expected masses and are more
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         25


 capability under LTQ 2.6 instrument control software with developer’s kit. Benzoic acid
                                                                                                                                                                                                               6 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    25
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 z32+3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              c10+1
                                                                                                                                                                                      Relative Abundance




                                                                                                                                                                                                               6 0                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    20
                                                                                                                                                                                                               5 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       15                                                                                                                                                                                                                           15



 anions which are generated in the chemical ionization source at the rear of the
                                                                                                                                                                                                               5 0
                                                                                                                                                                                                               4 5

                                                                                                                                                                                                               4 0
                                                                                                                                                                                                                                                                                                                                   c 7
                                                                                                                                                                                                                                                                                                                                         + 1

                                                                                                                                                                                                                                                                                                                                   7 7 4 .9

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              + 1                                                                                                                                                               c ions                  MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         10

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        5
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           easily interpreted using standard data analysis software.
                                                                                                                                                                                                               3 5                                                                           + 1                                                                                             + 1
                                                                                                                                                                                                                                                                                                                                                                                                                          + 1
                                                                                                                                                                                                                                                                                                                                                                                                                      z 1 2                                                                                              z 1 7                                                                                                                                                                                                                                                                                                                                                                                                                                         0
                                                                                                                                                                                                                                                                                          c 4                                                                                             c 1 2                                                                                                                          2 0 3 0 .4


 instrument were used as PTR reagent. The anion target was 2e5. Activation time was
                                                                                                                                                                                                                                                                                                                                                                                                                      1 4 2 1 .0          + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                       z 1 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               960           965             970            975            980                985                990                995             1000            1005                                                        1220            1225                               1230              1235           1240              1245            1250            1255           1260         1265
                                                                                                                                                                                                               3 0                                                                        4 7 4 .8                                                       + 1                              1 3 8 4 .0
                                                                                                                                                                                                                                                                                                                                                                                                                                       1 5 7 9 .3

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   .                                                                                                                                                                m/z
                                                                                                                                                                                                                                                                                                             + 2                                      z 7                                                                                                                                      + 1
                                                                                                                                                                                                                                                                     + 2                                  z 1 2                                                                                                                                                                                                                                                                                                            + 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              m/z

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Improved sensitivity, resolution, as well as faster scan rates in the LTQ Velos dual-
                                                                                                                                                                                                                                                                                                                                                                              + 2                                                                                                  c 1 8
                                                                                                                                                                                                                                                                   c 8                                                                                                  z 1 7                                                                                                                                                                                       z 2 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           + 1                                          z 2 2
                                                                                                                                                                                                               2 5                                                                                        7 1 0 .8                                    9 1 2 .0                                                                                                                     2 0 2 1 .4
                                                                                                                                                                                                                                                                   4 6 5 .6                                                                                             1 0 1 5 .3                                                                                                                                                                                                                                      2 5 5 9 .6

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG                                                                              z ions
                                                                                                                                                                                                                                                                                              + 1                                                                                                                                                                             + 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     z 1 8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          + 1                       2 4 5 8 .6
                                                                                                                                                                                                                                                                                           c 5                                                                                                                                                                              z 1 5
                                                                                                                                                                                                               2 0                                                                                                                                                                                                                                                          1 7 4 6 .2                                               2 1 0 1 .5                                                                             z 2 3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 + 1
                                                                                                                                                                                                                                                                                           6 0 1 .9                                                                     + 1




 5 - 10 msec for ETD or for 25 -50 msec for PTR.
                                                                                                                                                                                                                                                                                                                                                                     c 9
                                                                                                                                                                                                               1 5                                   + 1                                                                                                                                                                                                                                                                                                                                                                    2 7 1 5 .7
                                                                                                                                                                                                                                                   c 2                                                                                                               1 0 5 8 .2


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           pressure linear trap result in extensive sequence coverage of intact proteins of up to
                                                                                                                                                                                                                                                   2 4 4 .8
                                                                                                                                                                                                               1 0
                                                                                                                                                                                                                                         + 2
                                                                                                                                                                                                                                     c 3
                                                                                                                                                                                                                 5
                                                                                                                                                                                                                                     1 7 3 .7
                                                                                                                                                                                                                 0
                                                                                                                                                                                                                       0                                                              5 0 0                                                             1 0 0 0                                                        1 5 0 0                                                                         2 0 0 0                                                                                  2 5 0 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               SHHWGYGKHNGPEHWHKDFPIANGERQSPVDIDTKAVVQDPAL
                                                                                                                                                                                                                                                                                                                                                                                                                  m   /z
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           30kDa in a single 10 minute experiment.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               KPLALVYGEATSRRMVNNGHSFNVEYDDSQDKAVLKDGPLTGT
                                           High Pressure Cell
                                                                             Low Pressure Cell                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         LTQ Velos – 10 ms ETD with 25 ms PTR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                With product ions of +5, +6 and even +7 charge resolved in full zoom scan mode,
                                           High Trapping Efficiency
                                          High Dissociation Efficiency    Higher Resolution/Scan Rate                                                    •     For analysis on a unit resolution instrument charge reduction of product ion is desired                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         YRLVQFHFHWGSSDDQGSEHTVDRKKYAAELHLVHWNTKYGDF                                                                                                                                                                                                                                                                                                                                                                 LTQ Velos instrument identifies many c’/z. product ions as big as 10 kDa in the
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               c ions                   MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
                                           High Isolation Efficiency                                                                                           when precursor ion of high charge state are produced, as is the case in intact protein
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   .
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  z ions                                                                                       GTAAQQPDGLAVVGVFLKVGDANPALQKVLDALDSIKTKGKST                                                                                                                                                                                                                                                                                                                                                                 complex spectrum of intact myoglobin and carbonic anhydrase.
    Electrospray                                                                                                                                               analysis.
       Source
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               DFPNFDPGSLLPNVLDY WTYPGSLTTPPLLESVTWIVLKEPISVS                                                                                                                                                                                                                                                                                                                                                              The number of identified ions,and thus the sequence coverage for the protein is
                                                           Front            Center
                                                                                     Detector
                                                                                                Back                                                     •     Both extended ETD reaction and PTR can be used to reduce the product ion charge
                                                           Lens             Lens
                                                                                        1
                                                                                                Lens
                                                                                                                                                               state.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          SQQMLKFRTLNFNAEGEPELLMLANWRPAQPLKNRQVRGFPK                                                                                                                                                                                                                                                                                                                                                                  significantly improved in the LTQ Velos dual-pressure ion trap compared to the LTQ
                                                Octopole
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Extensive sequence coverage (identified c’/z. ion are colored) is obtained from spectral                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           XL ion trap.
                                                                                                                             e-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             •
                                                                    HPT
                                                                   HPC               LPC                                                                 •     Charge reduced product ions from extended ETD reaction times often contain extra
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        averaging a 1 min ETD with PTR experiment in both instruments.
                                                                                                                                                               hydrogen which cause a mass shift (insert). These ions are usually not assigned during                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      •              Extensive sequence coverage is obtained from averaging 10 min ETD with PTR
                                                                                     Detector
                                                                                                                                                               database searches (the colored peaks are ions identified by the database search).                                                                                                                                                                                                                                                                                                                                                                                                                                                             •          PTR reaction times are adjusted such that the reduced charge states of product ions                                                                                                                                                                               spectra in a single data acquisition experiment on the LTQ Velos instrument.
                                                                                       2
                                                                                                                                                               Extended ETD reaction times also generate internal fragments as noise peaks which                                                                                                                                                                                                                                                                                                                                                                                                                                                                        are resolved for the instrument used under full scan zoom mode, which is +1 to +3 for
                         S-lens
                    5 X More Sensitive                                                                  ETD Reagent Heated        PTR Reagent Heated           complicate the data analysis.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            the LTQ XLTM ion trap or +1 to +7 for the LTQ Velos dual-pressure ion trap.                                                                                                                                                                        •              A total of 155 or 187 c’/z. ions are indentified from intact myoglobin or carbonic
                   Lower Injection Time
                                                                                                               Inlet                     Inlet
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          anhydrase, respectively, corresponding to the sequence coverage shown above.
                                                                                                                                                         •     PTR, on the other hand, produces charge reduced product ions with expected masses                                                                                                                                                                                                                                                                                                                                                                                                                                                             •          The sequence coverage for the 1-min acquisition is significantly improved on the LTQ
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Under this experimental condition, the largest c’/z. product ions identified are c’96 of
                                                                                                                   N2 CI/Carrier Gas

                                                                                                                                                               for database searches. ETD-PTR spectra also contain less noise. Thus sequence                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Velos instrument due to the new technologies in this new dual-pressure linear trap,                                                                                                                                                                •
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           .                                                              .
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          10610.7 Da and z 94 of 10267.6 Da for myglobin, and c’83 of 9326.6 Da and z 87 of
                                                                                                                                                               coverage from ETD-PTR experiments is better than that from extended ETD                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  which resulted in improved sensitivity, resolution, as well as faster scan rate.
                                                                                                                                                               experiments (more colored peaks).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          9936.2 Da for carbonic anhydrase.                                                                                                                                                                                                                                                                                                                                               Picotip is a trademark of New Objective. All other trademarks are the property of Thermo Fisher Scientific and its
                           LTQ VelosTM ETD PTR Instrument Configuration
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          subsidiaries.

				
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