Abstract
AbstractOpen searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of post-translational modification profiles detected in open searches based on attributes, such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multiexperiment comparisons for studying changes in modification profiles, e.g., in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed and paraffin-embedded samples, detects extreme underalkylation of cysteine in some data sets, discovers an artifactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multicenter proteomics profiling study.
Highlights
Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments
We demonstrate how post-translational modifications (PTMs)-Shepherd improves the analysis of data from formalin-fixed and paraffin-embedded samples, detects extreme underalkylation of cysteine in some data sets, discovers an artifactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multicenter proteomics profiling study
The process starts with PTM-Shepherd reading false discovery rate (FDR)-filtered peptide-spectrum match (PSM) lists (produced by MSFragger and Philosopher, optionally with open search artifacts removed using Crystal-C [24]), and the mass shift for each PSM, to construct a mass shift histogram [11]
Summary
Proteomics open searches require extensive post hoc analysis to be useful. PTMShepherd provides comprehensive open search annotation from peptidespectrum match lists, including modification ID, localization, and effects on spectral similarity (SS) and retention time (RT). We present PTM-Shepherd, a bioinformatics tool that automates characterization of post-translational modification profiles detected in open searches based on attributes, such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. Database searching of shotgun proteomics data is a commonly used strategy for identification of peptides and proteins from complex protein mixtures [1, 2] Peptide identification in this strategy most commonly relies on matching tandem mass spectrometry (MS/MS)–derived peptide spectra to their theoretical counterparts using MS/MS database search tools, which require prior knowledge of the potential modifications that might be present in a sample. We present PTM-Shepherd, an automated tool that calls modifications from open search PSM lists and characterizes them based on attributes, such as amino acid localization, fragmentation spectra similarity, effect on RT, and relative modification rates. PSMs are assigned to the peak if their mass shift falls within the reported peak boundary
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