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m a s s s p e c t r o m e t r y P R O D U C T S U P P O R T B U L L E T I N 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 95 90 Precursor Charge State 85 ETD spectra usually contain undisso- 80 75 ciated precursor ions as charge- 70 MH3+ MH2+ reduced precursor peaks that can +1 65 c 11 1070.3 provide information about the charge 60 MH1+ state of the precursor ion. In most Relative Abundance 55 50 45 +1 +1 z4 434.1 z8 +1 cases, these charge-reduced precursor c3 805.0 40 309.2 z5 +1 c7 +1 +1 +1 z 11 ion peaks are the most intense peaks c8 +1 35 30 c4 +1 491.1 699.3 814.2 +1 +1 1080.2 c 14 1373.2 in the ETD spectrum, and the 424.2 z9 c 10 +1 z 25 +1 z6 619.2 +1 z7 690.1 876.1 1013.2 +1 z 10 12 1195.2 precursor charge state information is +1 933.1 20 15 z3 363.1 c +1 5 571.3 c 12 +1 readily obtained. Sometimes, not all +1 +1 c2 1141.4 10 +1 y1 z2 232.0 252.2 the charge-reduced precursor ion 5 0 147.1 peaks are present or they are not the 0 200 400 600 800 m/z 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 0 +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 8000 P ro c e s s e d work efﬁciently, the .SRF ﬁle format must 6000 be used for the database search. Alternat- 4000 ively, for proper searching of ETD data with 2000 MASCOT, ETD data pre-processing in 0 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) www.thermo.com
"Efficient Data Pre-processing of Unit Resolution ETD MSMS Spectra"