PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline

Abstract

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms and demonstrate its performance on ground-truth synthetic peptide reference datasets, one previously published small dataset, one new larger dataset, and also on a previously published phosphoenriched dataset where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

“…Using the PTMProphet tool (50) which is built into FragPipe and has recently been tailored to localize any assigned modifications, we assessed the probability score for localization and found that for most peptides the localization probability is greater than 0.8 (Figure 3E).…”

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

“…Existing tools for open search postprocessing perform a limited set of analyses on a spectrumlevel basis. PTM-Prophet (13), for example, is limited to localizing mass differences for each PSM but does not provide data summaries that can inform subsequent searches nor does it provide identities for mass differences. Philosopher (14) only provides mappings of mass differences to UniMod and generates a basic mass shift histogram.…”

SIMSI-Transfer: Software-Assisted Reduction of Missing Values in Phosphoproteomic and Proteomic Isobaric Labeling Data Using Tandem Mass Spectrum Clustering

“…When only a few (if any) fragment ions can be observed in MS2 spectra that distinguish the different localization possibilities, it becomes increasingly more difficult to locate the modification site accurately (26). It should be noted that database search algorithms such as Andromeda from MaxQuant and Mascot by Matrix Science also suffer from this issue (27), and other more dedicated software packages can be used to resolve such ambiguities at least partially (28)(29)(30)(31). We chose to retain such ambiguous phosphopeptide spectra clusters because we deem these to contain valuable information for further biology-centered data analysis.…”