Efficient Data Pre-processing of Unit Resolution ETD MSMS Spectra by maclaren1


									    m a s s                                s p e c t r o m e t r y

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                                                                              Efficient Data Pre-processing of
          PSB 125                                                             Unit Resolution ETD MS/MS Spectra
          Page 1 of 2
                                                                              Confident assignment of the charge state of a precursor from which a particular MS/MS
                                                                              spectrum is derived is crucial for successful and efficient spectrum analysis. High quality
                                                                              spectra obtained by Electron Transfer Dissociation (ETD) are usually observed from peptide
                                                                              precursors with charge states 3+ and higher. In the absence of precursor ion charge state
                                                                              information from unit resolution full MS spectra, several charge states are assumed for each
                                                                              precursor ion when a database search is performed. Therefore, uncertainty about the precursor
                                                                              ion charge state of an ETD spectrum requires that each spectrum be searched several times
                                                                              in order to cover several potential precursor charge states (usually from 2+ to 7+).
                                                                                   A new ETD data pre-processing function was developed to automatically assign
                                                                              charge states to precursor ions according to the characteristics of ETD spectra, and is now
                                                                              included in BioWorks™ software version 3.3.1. Prior to a database search, the pre-
                                                                              processing function reads, extracts and examines the characteristics of an ETD spectrum in
                                                                              order to efficiently determine the precursor ion charge state. For example, one of the most
                                                                              important features of ETD spectra is the series of peaks from undissociated charge-reduced
                                                                              precursor ions. These characteristics and other spectral features, such as loss of NH2 from
                                                                              the charge-reduced precursors, provide information on the precursor ion charge state.
                                                                              Because the ETD spectrum pre-processing routine assigns a single charge state to each
                                                                              spectrum, an ETD spectrum no longer needs to be searched multiple times, which reduces
                                                                              not only the number of data files searched but also the number of false positive identifica-
                                                                              tions generated from multiple searches of each ETD spectrum.

                                 100                                                                                                                                                                           Determination of a Single
                                                                                                                                                                                                               Precursor Charge State
                                  85                                                                                                                                                                           ETD spectra usually contain undisso-
                                                                                                                                                                                                               ciated precursor ions as charge-
                                  70                                                      MH3+                        MH2+                                                                                     reduced precursor peaks that can
                                  65                                                                                                                         c 11
                                                                                                                                                             1070.3                                            provide information about the charge
                                                                                                                                                                                                   MH1+        state of the precursor ion. In most
            Relative Abundance

                                  45                                     +1
                                                                                                                                                                                                               cases, these charge-reduced precursor
                                                                        c3                                                        805.0
                                                                                                                                                             z 11
                                                                                                                                                                                                               ion peaks are the most intense peaks
                                                                                                                                     c8                                                +1

                                                                                           491.1                      699.3
                                                                                                                                               +1    +1
                                                                                                                                                             1080.2                   c 14
                                                                                                                                                                                      1373.2                   in the ETD spectrum, and the
                                                                              424.2                                                          z9       c 10                      +1
                                                                                                                                             876.1 1013.2
                                                                                                                                                 z 10
                                                                                                                                                                             1195.2                            precursor charge state information is
                                                                              +1                                                                 933.1
                                                                                                    571.3                                                      c 12
                                                                                                                                                                    +1                                         readily obtained. Sometimes, not all
                                                     +1          +1
                                                                c2                                                                                             1141.4
                                  10         +1
                                                    232.0       252.2                                                                                                                                          the charge-reduced precursor ion
                                                                                                                                                                                                               peaks are present or they are not the
                                       0                  200                      400                    600                  800
                                                                                                                                                    1000                 1200                   1400
                                                                                                                                                                                                               most intense peaks. In this case,
                                                                                                                                                                                                               additional spectral characteristics are
                                           Figure 1. ETD spectrum characteristics which provides information                                                                                                   used to determine the correct
                                                     for single precursor charge state determination                                                                                                           precursor charge state.

Part of Thermo Fisher Scientific
                                                                   P R O D U C T                         S U P P O R T   B U L L E T I N    1 2 5

              Efficient Data Pre-processing of
              Unit Resolution ETD MS/MS Spectra
Page 2 of 2
              ETD Data Pre-processing Using BioWorks 3.3.1 Software – Charge State Assignment
              All ETD spectra are extracted from the raw file with a default
              charge state of 2+, and the correct precursor charge state
              assignment is subsequently updated in the DTA information
              panel, as illustrated in Figure 2.
                       The distribution of precursor charge states after the ETD
              spectra pre-processing step is shown in Figure 3. Given the
              number of spectra from precursors with charge states > 3+, a
              significant number of spectra would have been missed by using
              the automatic generation of 2+ and 3+ for data analysis. As
              demonstrated by experimental data (not shown here), the pre-
                                                                                                                    Before                  After
              processing function creates a single correct charge state per
              spectrum an average of 90% of the time. For the remaining                                             Figure 2: DTA table in BioWorks
              10% of the spectra, the two most likely charge states are                                            before and after precursor charge
                                                                                                                           state assignment
              assigned, and in most cases, one of the two charge states
              assigned is the correct one.
                                    Distribution of Precursor Charge State
                                                                                                         The pre-processing function was
                2 50 0                                                                              performed on several data sets of different
                                                                                                    protein digests and the results are displayed
                2 00 0
                                                                                                    in Figure 4. Pre-processing of the ETD
              Number of Spectra

                1 50 0                                                               da t a s e t 1 spectra reduced the overall number of files
                                                                                     da t a s e t 2

                1 00 0                                                               da t a s e t 3 for database searching by greater than a
                                                                                                    factor of five. Therefore, database search
                  50 0
                                                                                                    time was significantly reduced. Without this
                          +2            +3        +4          +5         +6       +7
                                                                                                    processing function, researchers would
                                               Charge State                                         either have to rely on the automatic
                       Figure 3: Distribution of Precursor Charge State                             generation of 2+ and 3+ precursor ions, thus
                                 Data Set 1: 9-protein LysC digest                                  missing all of the precursor ions of charge
                           Data Set 2: 9-protein digest (at K and R)
                   Data Set 3: ABRF sPRG 49-protein digest (at K and R)                             states above 3+, or have to search five times
                                                                                                    more spectra to capture all the relevant
                                Number of Spectra to be Searched                                    information in the data set.
                                  180 00                                                                 The ETD data pre-processing function
                                  160 00
                                                                                                    is enabled by default for database searches
                                  140 00
                                                                                                    of ETD spectra in BioWorks 3.3.1 software.
              Number of Spectra

                                  120 00

                                  100 00
                                                                                                    Note that for ETD data pre-processing to
                                                                                N o P r o c e s s in g

                                                                                P ro c e s s e d    work efficiently, the .SRF file format must
                                   6000                                                             be used for the database search. Alternat-
                                                                                                    ively, for proper searching of ETD data with
                                                                                                    MASCOT, ETD data pre-processing in
                             data set 1            data set 2          data set 3                   BioWorks 3.3.1 must be used before data
                        Figure 4: Reduction of the Number of Spectra                                export to MASCOT in mzData format.
                                                 Needed to be Searched
                                             Data Set 1: 9-protein LysC digest
                                         Data Set 2: 9-protein digest (at K and R)
                                   Data Set 3: ABRF sPRG 49-protein digest (at K and R)


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