Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation

Abstract

Mass spectrometry-based chemoproteomics has enabled functional analysis and small molecule screening at thousands of cysteine residues in parallel. Widely adopted chemoproteomic sample preparation workflows rely on the use of pan cysteine-reactive probes such as iodoacetamide alkyne combined with biotinylation via copper-catalyzed azide–alkyne cycloaddition (CuAAC) or “click chemistry” for cysteine capture. Despite considerable advances in both sample preparation and analytical platforms, current techniques only sample a small fraction of all cysteines encoded in the human proteome. Extending the recently introduced labile mode of the MSFragger search engine, here we report an in-depth analysis of cysteine biotinylation via click chemistry (CBCC) reagent gas-phase fragmentation during MS/MS analysis. We find that CBCC conjugates produce both known and novel diagnostic fragments and peptide remainder ions. Among these species, we identified a candidate signature ion for CBCC peptides, the cyclic oxonium-biotin fragment ion that is generated upon fragmentation of the N­(triazole)–C­(alkyl) bond. Guided by our empirical comparison of fragmentation patterns of six CBCC reagent combinations, we achieved enhanced coverage of cysteine-labeled peptides. Implementation of labile searches afforded unique PSMs and provides a roadmap for the utility of such searches in enhancing chemoproteomic peptide coverage.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

“…Here we envisioned combining proximity labeling via the ultra-fast biotin ligase TurboID with cysteine chemoproteomics 11,21,[24][25][26][27][28][29][30]57,58 to enable fractionation-free capture of the subcellular cysteinome, for both residue identification and quantification of cysteine oxidation. We were inspired by recent reports of two-step capture for subcellular phosphoproteomics, in which proteins biotinylated by TurboID were first enriched on avidin resin followed by peptide-level capture of phosphopeptides 59 .…”

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

“…Chemoproteomic studies, including our own 15,17,47,48,[53][54][55] , continue to rely on stable isotope incorporation, either through metabolic labeling or the aforementioned custom isotopically labeled capture reagents, and MS1 based quantification 10,42,[56][57][58] . These methods are widely adopted in large part due to their relatively reasonable cost, the compatibility of the resulting datasets with freely available software packages for analysis [59][60][61] , and increased data reproducibility afforded by the ability to combine 'treated' and 'control' samples early in the sample preparation workflow.…”

“…For azide source, we chose to compare ß-azidoalanine with azidolysine with the goals of determining how reducing the reagent size would impact proteomic coverage and click efficiency, together with assessing how triazole fragmentation would be impacted by choice of azide. Our prior study 55 had revealed that the gas phase fragmentation of triazole modified peptides produced signature fragment ions, with the identity, intensity, and specificity of the produced ions varying depending on the nature of the azide. Therefore, we speculated that we might observe differences in the fragmentation pattern of azidohomoalanine and azidolysine based reagents, which could be harnessed for the production of a novel isobaric labeling strategy.…”

“…Motivated by our recent use of diagnostic feature detection 73 to identify characteristic fragment ions derived from biotin-modified precursor ions 55 , we next subjected our sCLIP datasets to diagnostic feature extraction using FragPipe with MSFragger and PTM-Shepherd (Figure 4A).…”

“…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.…”