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

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.