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Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator.

Wright JC, Collins MO, Yu L, Käll L, Brosch M, Choudhary JS - Mol. Cell Proteomics (2012)

Bottom Line: We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data.Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%).We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

View Article: PubMed Central - PubMed

Affiliation: Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Hinxton, Cambridge.

ABSTRACT
Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

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E. coli sequential experiment spectral pair analysis—This figure provides an in-depth breakdown of the spectral pairs in the E. coli partial tryptic digest sequential data set. A, This table shows the ratio of spectral pairs identified significantly by CID and ETcaD, CID only, and ETcaD only at a 0.01 q-value threshold across the range of precursor charge states. B, This chart shows the number of spectral pairs where the CID spectra or the ETcaD has the best score. For Mascot Percolator the PEP is used, for Mascot the ion score is used and for OMSSA the e-value is used. This data is displayed for all the spectral pairs with a match above the q-value threshold and also for only the spectral pairs where both CID and ETcaD PSMs are above the q-value threshold.
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Figure 7: E. coli sequential experiment spectral pair analysis—This figure provides an in-depth breakdown of the spectral pairs in the E. coli partial tryptic digest sequential data set. A, This table shows the ratio of spectral pairs identified significantly by CID and ETcaD, CID only, and ETcaD only at a 0.01 q-value threshold across the range of precursor charge states. B, This chart shows the number of spectral pairs where the CID spectra or the ETcaD has the best score. For Mascot Percolator the PEP is used, for Mascot the ion score is used and for OMSSA the e-value is used. This data is displayed for all the spectral pairs with a match above the q-value threshold and also for only the spectral pairs where both CID and ETcaD PSMs are above the q-value threshold.

Mentions: Fig. 7 contains an expanded analysis of the sequential E. coli experiment for each precursor, the CID and ETcaD fragmentation spectral pairs are directly compared. Fig. 7A depicts the performance of each search method; these have been further divided up by precursor charge state. As has been seen throughout this study, Mascot Percolator enhances the number of PSMs for both CID and ETcaD significantly identifying 8723 spectral pairs compared with 7178 for Mascot and 6689 for OMSSA. The total overlap in spectral pairs identified by both CID and ETcaD at a 0.01 q-value threshold covers 83% of the total significant spectral matched pairs for Mascot Percolator compared with 69 and 39% for Mascot and OMSSA. The fragmentation and charge bias seen in Mascot and OMSSA is less prominent with Mascot Percolator. In particular an extended overlap in identified CID/ETcaD spectral pairs for Mascot Percolator, where triply charged PSMs show 88% coincidence and 66% for PSMs with >3+ charge states, compared with 77 and 42% for Mascot, and 70 and 50% for OMSSA. Mascot and OMSSA significantly identify 50 and 57% of the 3+ charged CID spectra in the identified spectral pairs respectively, using Mascot Percolator this increases to 70%. Reciprocally, Mascot Percolator finds 90% of the 2+ identified spectral pairs to have significant ETcaD spectra compared with 81% for Mascot and only 6% for OMSSA.


Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator.

Wright JC, Collins MO, Yu L, Käll L, Brosch M, Choudhary JS - Mol. Cell Proteomics (2012)

E. coli sequential experiment spectral pair analysis—This figure provides an in-depth breakdown of the spectral pairs in the E. coli partial tryptic digest sequential data set. A, This table shows the ratio of spectral pairs identified significantly by CID and ETcaD, CID only, and ETcaD only at a 0.01 q-value threshold across the range of precursor charge states. B, This chart shows the number of spectral pairs where the CID spectra or the ETcaD has the best score. For Mascot Percolator the PEP is used, for Mascot the ion score is used and for OMSSA the e-value is used. This data is displayed for all the spectral pairs with a match above the q-value threshold and also for only the spectral pairs where both CID and ETcaD PSMs are above the q-value threshold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412976&req=5

Figure 7: E. coli sequential experiment spectral pair analysis—This figure provides an in-depth breakdown of the spectral pairs in the E. coli partial tryptic digest sequential data set. A, This table shows the ratio of spectral pairs identified significantly by CID and ETcaD, CID only, and ETcaD only at a 0.01 q-value threshold across the range of precursor charge states. B, This chart shows the number of spectral pairs where the CID spectra or the ETcaD has the best score. For Mascot Percolator the PEP is used, for Mascot the ion score is used and for OMSSA the e-value is used. This data is displayed for all the spectral pairs with a match above the q-value threshold and also for only the spectral pairs where both CID and ETcaD PSMs are above the q-value threshold.
Mentions: Fig. 7 contains an expanded analysis of the sequential E. coli experiment for each precursor, the CID and ETcaD fragmentation spectral pairs are directly compared. Fig. 7A depicts the performance of each search method; these have been further divided up by precursor charge state. As has been seen throughout this study, Mascot Percolator enhances the number of PSMs for both CID and ETcaD significantly identifying 8723 spectral pairs compared with 7178 for Mascot and 6689 for OMSSA. The total overlap in spectral pairs identified by both CID and ETcaD at a 0.01 q-value threshold covers 83% of the total significant spectral matched pairs for Mascot Percolator compared with 69 and 39% for Mascot and OMSSA. The fragmentation and charge bias seen in Mascot and OMSSA is less prominent with Mascot Percolator. In particular an extended overlap in identified CID/ETcaD spectral pairs for Mascot Percolator, where triply charged PSMs show 88% coincidence and 66% for PSMs with >3+ charge states, compared with 77 and 42% for Mascot, and 70 and 50% for OMSSA. Mascot and OMSSA significantly identify 50 and 57% of the 3+ charged CID spectra in the identified spectral pairs respectively, using Mascot Percolator this increases to 70%. Reciprocally, Mascot Percolator finds 90% of the 2+ identified spectral pairs to have significant ETcaD spectra compared with 81% for Mascot and only 6% for OMSSA.

Bottom Line: We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data.Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%).We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

View Article: PubMed Central - PubMed

Affiliation: Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Hinxton, Cambridge.

ABSTRACT
Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

Show MeSH
Related in: MedlinePlus