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

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 data sets, one previously published small
data set, one new larger data set, and also on a previously published
phosphoenriched data set where the correct sites of modification are
unknown. Data have been deposited to ProteomeXchange with identifier
PXD013210.