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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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Related in: MedlinePlus

E. coli peptide spectrum match q-p plots—These four q-value PSM plots display the estimated number of correct PSMs for the partial E. coli digest data sets using Mascot, OMSSA, and Mascot Percolator across a range of q-value thresholds. Plot A, shows all the complete data sets and plots B, C, and D, show the estimated correct PSMs for 2+, 3+, and >3+ precursor charge states.
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Figure 5: E. coli peptide spectrum match q-p plots—These four q-value PSM plots display the estimated number of correct PSMs for the partial E. coli digest data sets using Mascot, OMSSA, and Mascot Percolator across a range of q-value thresholds. Plot A, shows all the complete data sets and plots B, C, and D, show the estimated correct PSMs for 2+, 3+, and >3+ precursor charge states.

Mentions: To alleviate the bias toward doubly charged precursors a partial tryptic digest was performed, the increased number of missed cleavages generates higher charged peptides. Interrogation of this data set reflects that 47% of the spectra have a precursor charge state greater than 2+. Fig. 5 displays q-value PSM plots for the parallel CID and ETcaD data sets, it shows that Mascot and OMSSA have a very similar performance for this data set, the only significant deviation being the doubly charged OMSSA ETcaD PSMs. Mascot Percolator performs well at all charge states for both CID and ETcaD data, resulting in a 28% CID and 171% ETcaD increase in PSMs compared with OMSSA and a 32% CID and 36% ETcaD increase with respect to Mascot (Table 4B). A list of peptides uniquely identified by Mascot Percolator for the E. coli CID and ETcaD data sets is provided in the supplementary material.


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 peptide spectrum match q-p plots—These four q-value PSM plots display the estimated number of correct PSMs for the partial E. coli digest data sets using Mascot, OMSSA, and Mascot Percolator across a range of q-value thresholds. Plot A, shows all the complete data sets and plots B, C, and D, show the estimated correct PSMs for 2+, 3+, and >3+ precursor charge states.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: E. coli peptide spectrum match q-p plots—These four q-value PSM plots display the estimated number of correct PSMs for the partial E. coli digest data sets using Mascot, OMSSA, and Mascot Percolator across a range of q-value thresholds. Plot A, shows all the complete data sets and plots B, C, and D, show the estimated correct PSMs for 2+, 3+, and >3+ precursor charge states.
Mentions: To alleviate the bias toward doubly charged precursors a partial tryptic digest was performed, the increased number of missed cleavages generates higher charged peptides. Interrogation of this data set reflects that 47% of the spectra have a precursor charge state greater than 2+. Fig. 5 displays q-value PSM plots for the parallel CID and ETcaD data sets, it shows that Mascot and OMSSA have a very similar performance for this data set, the only significant deviation being the doubly charged OMSSA ETcaD PSMs. Mascot Percolator performs well at all charge states for both CID and ETcaD data, resulting in a 28% CID and 171% ETcaD increase in PSMs compared with OMSSA and a 32% CID and 36% ETcaD increase with respect to Mascot (Table 4B). A list of peptides uniquely identified by Mascot Percolator for the E. coli CID and ETcaD data sets is provided in the supplementary material.

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