Mining for ions: diagnostic feature detection in MS/MS spectra of post-translationally modified peptides

Abstract

Post-translational modifications (PTMs) are an area of great interest in proteomics, with a surge in methods to detect them in recent years. However, PTMs can introduce complexity into proteomics searches by fragmenting in unexpected ways. Detecting post-translational modifications in mass spectrometry-based proteomics traditionally relies on identifying ions shifted by the masses of the modifications. This presents challenges for many PTMs. Labile PTMs lose part of their modification mass during fragmentation, rendering shifted fragment ions unidentifiable, and isobaric PTMs are indistinguishable by mass, requiring other diagnostic ions for disambiguation. Furthermore, even modifications that have undergone extensive characterization often produce different fragmentation patterns across instruments and conditions. To address these deficiencies and facilitate the next generation of PTM identification, we have developed a method to automatically find diagnostic spectral features for any PTM, allowing subsequent searches to take advantage of additional metrics and increase PTM identification and localization rates. The method has been incorporated into the open-search annotation tool PTM-Shepherd and the FragPipe computational platform.

MSFragger-Labile: A Flexible Method to Improve Labile PTM Analysis in Proteomics

“…Fully enzymatic MSFragger search was performed using strict-trypsin settings (i.e., allowing cleavage before Pro) and allowing 2 missed cleavages, precursor and fragment tolerances of 20 and 10 ppm respectively, mass calibration, fixed Cys carbamidomethylation, and variable modifications of Met oxidation and Protein N-terminal acetylation. 4SU crosslinking was searched as both the intact nucleoside (+226.0594 Da) and the base only (+94.0168 Da), as frequent in-source fragmentation of nucleoside was noted by Bae et al and which we confirmed using the PTM-Shepherd diagnostic mining module (23). For labile search, +94 and +226 were searched as mass offsets on any amino acid, with labile mode active, no diagnostic or peptide remainder ions, and a fragment remainder ion of +94.…”

“…With user-specified options for diagnostic ions, peptide remainder masses, and fragment remainder masses, a wide variety of labile modifications can be easily searched with MSFragger. The labile mode search can leverage the fragment ions discovered by our recently described fragment ion discovery module in PTM-Shepherd (23), allowing labile mode to be used without requiring manual annotation of fragmentation pathways for new modifications. Localization of labile modifications has long been a significant obstacle to PTM analyses, and remains so in cases where PTMs dissociate without forming any fragment remainder masses.…”

“…Many modifications are not lost so cleanly, however, either leaving behind a piece of the modification, or taking a piece of the peptide with them when leaving, such as the net loss of water from phospho-Ser and Thr residues during neutral loss of phosphoric acid. Modification loss can also produce signature, or diagnostic, fragment ions in the mass spectrum that indicate the presence of a particular modification (23).…”

“…Here, we demonstrate the utility of labile mode searches to increase the spectral annotation rate of data for several labile modifications, including phosphorylation, peptide-RNA crosslinks, and ADP-ribosylation. We used our recently described diagnostic ion mining module in PTM-Shepherd to determine the fragment ions to use in labile search, allowing optimal settings to be chosen even for modifications without well characterized fragmentation pathways (23). Labile search has been incorporated into MSFragger starting with version 3.5 and FragPipe version 18.0, and workflow templates for the labile phosphorylation and ADP-ribosylation searches described here are available from the workflows menu in FragPipe.…”

A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

“…FragPipe with MSFragger labile search 72 and PTM-Shepherd diagnostic feature detection 73 revealed a high intensity and highly specific dihydrooxazolium ion unique to a subset of sCLIP reagents. By harnessing this dihydrooxazolium ion, we developed the sCLIP isobaric platform, which is a low cost six-plex isobaric labeling strategy in which the mass balancer and reporter are incorporated into cysteine-reactive iodoacetamide alkyne probe and sCLIP capture reagent, respectively (Figure 1C).…”