Modification Site Localization Scoring Integrated into a Search Engine

Peter R Baker,Robert J Chalkley,Jonathan C Trinidad

doi:10.1074/mcp.m111.008078

Peter R Baker, Robert J Chalkley + Show 1 more

Open Access

https://doi.org/10.1074/mcp.m111.008078

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Abstract

Large proteomic data sets identifying hundreds or thousands of modified peptides are becoming increasingly common in the literature. Several methods for assessing the reliability of peptide identifications both at the individual peptide or data set level have become established. However, tools for measuring the confidence of modification site assignments are sparse and are not often employed. A few tools for estimating phosphorylation site assignment reliabilities have been developed, but these are not integral to a search engine, so require a particular search engine output for a second step of processing. They may also require use of a particular fragmentation method and are mostly only applicable for phosphorylation analysis, rather than post-translational modifications analysis in general. In this study, we present the performance of site assignment scoring that is directly integrated into the search engine Protein Prospector, which allows site assignment reliability to be automatically reported for all modifications present in an identified peptide. It clearly indicates when a site assignment is ambiguous (and if so, between which residues), and reports an assignment score that can be translated into a reliability measure for individual site assignments.

Highlights

Large proteomic datasets identifying thousands of modified peptides are becoming increasingly common in the literature
E-values are calculated for each saved hit and converted into -10log10E The difference in these log evalues is reported for each site assignment as a SLIP (Site Localization In Peptide) score
Ascore threshold can be defined below which a site is defined as ambiguous

Summary

Introduction

Large proteomic datasets identifying thousands of modified peptides are becoming increasingly common in the literature. For the top hits saved for each spectrum Protein Prospector’s Batch-Tag calculates the scores for all permutations of the site assignments. The best hits with different site assignments are saved. E-values are calculated for each saved hit and converted into -10log10E The difference in these log evalues is reported for each site assignment as a SLIP (Site Localization In Peptide) score.

Results

Conclusion