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

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.