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

Abstract

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 PTM 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 multi-experiment 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 paraffin-embedded samples, detects extreme underalkylation of cysteine in some datasets, discovers an artefactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multi-center proteomics profiling study.

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome

“…Moreover, proteomic strategies used for mapping ADPr on glutamate and aspartate are based upon derivatization of ADPr into a chemical mark (+15.011 Da) using hydroxylamine (Zhang et al, 2013). However, use of hydroxylamine can induce chemical artifacts (+15.011 Da) on acidic residues (Geiszler et al, 2020), mimicking the mark used for ADPr identification. Indeed, treatment of bovine serum albumin with hydroxylamine under native conditions revealed the same aberrant chemical marks (+15.011 Da), which could lead to erroneous identification of ADPr-modified aspartate and glutamate residues (Supplementary Note).…”

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

“…In the first step, we optimised FragPipe's Open Search workflow to identify modified peptides and assign the masses of their respective modifications. For this purpose, we performed an Open Search using MSfragger 6,7,27,28 followed by removal of mass shift artifacts with The data is represented as letter plots. The size of the letter is scaled by the fraction of all modified sites that is modified at the indicated amino acid.…”

“…Crystal-C, 27 processing of peptide and protein identifications using PeptideProphet 29 and ProteinProphet, 30 false discovery-rate (FDR) filtering via Philosopher 31 and summarisation of the mass shifts observed on peptides using PTM-Shepherd. 28 Using this procedure, we identified the expected alkylation with IA-alkyne as the main modification (m exp ) in the form of two peaks corresponding to the isotopically differentiated isoDTB tags ( Fig. 2a,b).…”

Proteome-wide profiling and mapping of post translational modifications in human hearts

“…By applying the annotation tools PTM-sticker 11 and PTM-Shepherd 37 we were able to annotate the majority of the modification mass shifts identified and localized with a high accuracy. However, for approximately 15% of the mass shifts, we could not assign a known modification from the Unimod 38 database.…”

“…All the results of PTM-Sticker are reported in the Supplementary Tables S2 , S3 and S4 . In addition we used PTM-Shepherd 37 to confirm the PTM annotation from PTM-Sticker and to add additional information on the unknown masses.…”