Comparing Chromatography Columns for the Separation and ESI LC-MS by ida17629

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									  HUPO 2005




Comparing Chromatography Columns for the Separation and
ESI LC-MS/MS Analysis of Peptides and Protein Mixtures
Hansjörg Toll1, Ulrike Schweiger-Hufnagel2, Dirk Wunderlich2, Catherine Stacey2, Christian G. Huber1; 1Instrumental Analysis and Bioanalysis, Saarland
University, Saarbrücken, Germany; 2Bruker Daltonik GmbH, Bremen, Germany

Introduction                                                                 Results for Peptides                                                Results for Proteins
                                                                             10 tryptically digested proteins were separated on two              For analyzing the detection sensitivity due to different tuning
For ESI MS used for proteomic research, reversed-phase
                                                                             monolithic and a microparticular column, resp., and the MS          parameter sets (Cyt C and Lys tuning), the signal-to-noise
high-performance liquid chromatography (RP HPLC) is of
                                                                             data were compared in respect of:                                   ratios    for   eight   proteins   were    determined       from
outstanding relevance. This combination has made an
                                                                                                                                                 chromatographic peaks, revealing significant differences in the
enormous progress in the last decades: RP HPLC has                           Chromatogram Quality                                                detectability (Figure 4). Moreover, spectrum quality also
become more and more sensitive due to separation                             As shown in Figure 1, the 100 μm monolithic column showed           depended significantly on the tuning parameters. With Lys
performance and miniaturization while ESI MS has become                      the best separation, with slightly better patterning than the 200   tuning, which was optimized for highest sensitivity in the range
faster and more sensitive.                                                   μm monolithic column and significantly better separation than       of 1500-3000 m/z, all eight proteins were detected. With the
This poster shows an enhancement of separation technique.                    the microparticular column, where individual peaks were             Cyt C tuning, on the other hand, myoglobin, carbonic
Monolithic columns based on poly-(styrene-divinylbenzene)                    sitting on top of a broad hump of unresolved background.            anhydrase and cytochrome C were detected with significantly
(PSDB) with a new very narrow inner diameter are compared
                                                                                                                                                 higher sensitivity, while other proteins such as lysozyme or
to standard microparticular columns. The progress in column                  Cumulative Ion Score                                                transferrin remained undetected. As a consequence, it is
condition has a positive effect on the complete instrumental                 Figure 2 summarizes the average cumulative ion scores               strongly advisable to tune the mass spectrometer with a series
setup.                                                                       extracted from triplicate analyses. For all four different          of proteins with different physico-chemical properties and to
                                                                             concentrations the highest score was achieved using the 100         run analyses with these different tunings in order to achieve
Method                                                                       μm monolithic column due to the highest number of identified        maximum detection sensitivity for RP-HPLC-MS analysis of
Peptide analysis                                                             peptides and the highest individual ion scores.                     complex protein mixtures.
A mixture of 10 commercially available proteins was digested                 Peak Volume
with trypsin according to standard protocols. Four different                 The peak volume which depends on column diameter and                                                        1.5




                                                                                                                                                                                     ]
amounts (Table 2) were injected onto three different columns:                                                                                                                                      Cyt C Tuning




                                                                                                                                                                        .10-7 [counts
                                                                             peak dispersion was determined for 15 peptide peaks and                                                                                                               Cah
•PSDB based monolithic column 60 x 0.2 mm i.d.                               averaged. For monolithic columns (19.1 s and 19.4 s) it was
•PSDB based monolithic column 60 x 0.1 mm i.d.                               17 % smaller than for the microparticular column (23.1 s)
•RP column with C18 silica particles 75 x 0.075 mm i.d.                      indicating a better separation performance of the monolithic                                                                                              Lac B
LC and MS conditions are summarized in Table 1.                              columns.                                                                                                                                                   Myo
                                                                                                                                                                                                                     Rib Cyt C
                                                                                                                                                                                          0
 Parameter /              75 µm i.d.     100 µm i.d.     200 µm i.d.         Reproducibility of Peptide Identification                                                                         0                       10                         20



                                                                                                                                                          signal intensity
 column                    Pepmap         Monolith         Monolith          To investigate the reproducibility of peptide identifications for                                                                                                    time [min]
 Instrument               HCT (Bruker) combined to Ultimate (Dionex)         all three separation columns, the number of peptides found                                                  4.0


                                                                                                                                                                     ]
                                                                                                                                                                                                   Lys Tuning


                                                                                                                                                        .10-6 [counts
                                                                                                                                                                                                                                        Trf
 Flow rate A              200 nL/min     500 nL/min      2000 nL/min         once, twice or three times in three consecutive runs was                                                                                                              Cah
 Injection volume           100 nL         250 nL          1000 nL           determined. As shown in Figure 3 the highest number of                                                                                                                Lac A
                                                                                                                                                                                                                                         Lac B
 Linear gradient B          5-60%          0-40%            0-40%            peptides that were identified in triplicate was achieved with the
 Gradient                            ACN in 0.05 % TFA                       100 µm i.d. monolithic column, 24 % more than with the 75 µm                                                                            Rib
 Column                                                                      i.d. microparticulate column, and 53 % more than with the 200                                                                                 Cyt C Lys     Myo
                                              55 °C
 temperature                                                                 µm i.d. monolith.                                                                                            0
 Spray gas              15 psi           15 psi             20 psi                                                                                                                             0                       10                        20
 Dry gas                                      4 L/min                         Summary/ Conclusion for Peptides                                                                                                                                   time [min]
 Spray voltage                               - 3500 V                         For peptides the 100 μm monolithic column                            Fig. 4. TIC MS of 8 proteins for Cyt C and Lys tuning.
 Ion charge control                    MS, MS/MS: 70.000                      turned out to be most suitable. This is explained
 Full scan range                           500-1500 m/z                       by the monolithic phase combined to a                                Summary/ Conclusion for Proteins
 MS/MS scan                               200 - 2000 m/z
                                                                              remarkably small inner column diameter, leading                      For proteins monolithic columns are suited very
 Active exclusion                 30 s, exclude after 1 spectrum
 Frag. Energy                                  1.5 V
                                                                              to sharp chromatographic peaks and high relative                     well, in particular when using multiple tuning
 No. Precursors                                  3
                                                                              concentration of the eluting analytes.                               parameter sets.
 Smart frag. ramp                           30 - 200 %                            1.2
                                                                                                      100 µm i.d.
  Table 1: Experimental LC and MS conditions
  A for constant linear flow velocity; B for comparable separation window
                                                                                                      Monolith                                                                   7000
                                                                             signal intensity
                                                                                10 -6 [counts]




                                                                                                                                                                                                                                        100 μ m monolith
                  amount                                 amount 1
 Protei                            amount 1/4 amount 1/2                                                                                                                         6000                                                   200 μ m monolith
        Organism      1/8                                 (fmol)
    n
                    (fmol)
                                     (fmol)     (fmol)                                                                                                                                                                                  75 μ m Pepmap
                                                                                                                                                  Cumulative Mascot Score




 CYT    horse    80               160             320         640                                                                                                                5000
 BSA bovine      37.5             75              150         300
                                                                              .




 ß LAC bovine    55               110             220         440
                                                                                                                                                                                 4000
 CAH bovine      34.5             69              138         276
 CAT    bovine   17.5             35              70          140                         0
                                                                                                                                                                                 3000
 LYS    chicken  70               140             280         560                                 0              10       20       30   40
 MYO horse       55               110             220         440                                                     time [min]
 RIB A bovine    75               150             300         600
                                                                                  1.2                                                                                            2000
 TRF    human    12.5             25              50          100                                     200 µm i.d.
 α LAC bovine    70               140             280         560                                                                                                                1000
                                                                                                      Monolith
                                                                             signal intensity
                                                                                 10 -6 [counts]




  Table 2: Amount of the analyzed protein digests. For
  loading the respective sample amounts on three different                                                                                                                                0
  columns with different injection volumes, the sample                                                                                                                                         amount 1           amount 1/2 amount 1/4 amount 1/8
  concentrations were adjusted accordingly.
                                                                                                                                                           Fig. 2. Average cumulative ion scores of peptides identified
  Protein analysis                                                                                                                                         upon analysis of amounts 1, 1/2, 1/4, 1/8.
                                                                               .




                                                                                                                                                                                                        triplicate         duplicate           single
  Parameter / tuning                 Lys tuning           Cyt C tuning                    0                                                                                                                                                    identification
                                                                                                                                                                                         140
                                 mixture of 8 proteins (same as in Table 2                        0              10       20       30   40
  Sample
                                 except for BSA and catalase)
                                                                                                                      time [min]                                                         120           21                   35
  Flow rate                                       2 ul/ min
                                                                                   1.0                                                                                                                                                             27
  Injection amount                         62.5 fmol per protein                                      75 µm i.d.
                                                                                                                                                                   Number of Peptides




                                                                                                                                                                                         100           21
  Linear gradient                       15-50% ACN in 0.05 % TFA
                                                                                                      Pepmap
                                                                             signal intensity




  Temperature                                       55 °C                                                                                                                                                                                          25
                                                                                 10 -6 [counts]




                                                                                                                                                                                          80                                 43
  Trap drive                     179.1               93.2
  Skimmer                        150.0 V             34.4 V
                                                                                                                                                                                          60
  Oct 1                          12 V                12 V
  Oct 2                          3.93 V              2.46 V                                                                                                                                            92
                                                                                                                                                                                          40
  Octopole RF Amplitude          167.2 Vpp           88.5 Vpp                                                                                                                                                                                      74
                                                                                                                                                                                                                             60
                                                                               .




  Lens 1                         - 1.7 V             - 1.9 V
                                                                                                                                                                                          20
  Lens 2                         - 31.1 V            - 36.1 V
  Cap exit                       340 V               253.8 V                               0                                                                                               0
  HV Capillary                                     3500 V                                         0              10       20       30   40                                                         100 μ m i.d.                                 75 μm i.d.
                                                                                                                                                                                                                       200 μm i.d.
  Dry gas                                         4 L/min                                                             time [min]                                                                   monolith                                     Pepmap
                                                                                                                                                                                                                       monolith
  Table 3: Sample, Experimental LC and MS conditions                              Fig. 1. TIC MS trace of the LC-MS/MS analysis of a peptide
                                                                                  mixture using monolithic and microparticular columns.                         Fig. 3. Reproducibility of peptide identifications for                                      three
                                                                                  Sample amount 1/8. Experimental conditions are given in                       consecutive analyses with amount 1/4.
                                                                                  Table 1.

								
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