PTM-Shepherd: Analysis and Summarization of Post-Translational and Chemical Modifications From Open Search Results

• Published in: Molecular & cellular proteomics Vol. 20; p. 100018
• Main Authors: Geiszler, Daniel J., Kong, Andy T., Avtonomov, Dmitry M., Yu, Fengchao, Leprevost, Felipe da Veiga, Nesvizhskii, Alexey I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2021
• Subjects: Open searching, PTM, Post-translational modification, Mass-tolerant search, Localization, Spectral similarity, Retention time; SS; CAA; RT; MS; BI; IAA; PSM; UCEC; Open searching, PTM, Post-translational modification, Mass-tolerant search, Localization, Spectral similarity, Retention time; LUAD; PTMs; EGS; QC; FDR; FFPE; JHU; SNR; PNNL; TMT; CPTAC; CCRCC; Tandem Mass Spectrometry; Post-translational modifications; Protein Identification; Bioinformatics; Quality control and metrics
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.TIR120.002216

Open 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. [Display omitted] •Comprehensive open-search annotation.•Sensitive modification detection.•Identification of batch effects.•Novel post-translational modification discovery. Proteomics open searches require extensive post hoc analysis to be useful. PTM-Shepherd provides comprehensive open search annotation from peptide-spectrum match lists, including modification ID, localization, and effects on spectral similarity (SS) and retention time (RT). These features can be used to find batch effect and new post-translational modifications, and inform subsequent closed searches. It is available as a standalone JAR and as part of the FragPipe suite.