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

Abstract

Post-translational modifications of proteins play essential roles in defining and regulating the functions of the proteins they decorate, making identification of these modifications critical to understanding biology and disease. Methods for enriching and analyzing a wide variety of biological and chemical modifications of proteins have been developed using mass spectrometry (MS)-based proteomics, largely relying on traditional database search methods to annotate resulting mass spectra of modified peptides. These database search methods treat modifications as static attachments of a mass to particular position in the peptide sequence, but many modifications undergo fragmentation in tandem MS experiments alongside, or instead of, the peptide backbone. While this fragmentation can confound traditional search methods, it also offers unique opportunities for improved searches that incorporate modification-specific fragment ions. Here, we present a new Labile Mode in the MSFragger search engine that can tailor modification-centric searches to the fragmentation observed. We show that labile mode can dramatically improve spectrum annotation rates of phosphopeptides, RNA-crosslinked peptides, and ADP-ribosylated peptides. Each of these modifications presents distinct fragmentation characteristics, showcasing the flexibility of MSFragger labile mode to improve search for a wide variety of biological and chemical modifications.

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).…”

“…Using the identified ions, we next re-searched our datasets using MSFragger labile ion search 55,72,82 to pinpoint ions that exhibited both high intensity and high (near 100%) frequency of detection in modified peptide spectra, while simultaneously having low background identification in unmodified peptide spectra. Exemplifying this process, for M1 precursors modified with reagent 12 (Figure 4B), we identified a characteristic ion with m/z 510.3762, which we ascribed to formation of the F1 ammonium ion through N26-C27 amide bond cleavage.…”

Cellular protein painting for structural and binding sites analysis via lysine reactivity profiling with o-phthalaldehyde

“…Lysine-reactive probes often react with proteins at low stoichiometry that necessitate enrichment to identify peptides containing the probe-modified lysine, while MS fragmentation could lead to probe cleavage generating daughter ions with unpredictable mass-to-charge ratio (m/z) (26). We hypothesized that these challenges can be circumvented by identifying lysine probes, preferably membrane-permeable, that interfere with the cleavage of proteins by trypsin at C-terminal of arginine or lysine.…”

Enhancing multiplexed cysteine chemoproteomics by uniting FragPipe with solid-phase compatible dialkoxydiphenylsilane reagents

“…FragPipe with MSFragger labile search 73 and PTM-Shepherd diagnostic feature detection 74 revealed a high intensity and highly specific dihydrooxazolium ion unique to a subset of sCLIP reagents; notably this ion forms as a result of gas-phase cleavage of the click-chemistry formed triazole moiety. 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 1B).…”

“…Using the identified ions, we next re-searched our datasets using MSFragger labile ion search 56,73,83 to pinpoint ions that exhibited both high intensity and high (near 100%) frequency of detection in modified peptide spectra, while simultaneously having low background identification in unmodified peptide spectra. Exemplifying this process, for M1 precursors modified with reagent 12 (Figure 4B), we identified a characteristic ion with m/z 510.3762, which we ascribed to formation of the F1 ammonium ion through N26-C27 amide bond cleavage.…”