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

Anal Chem. 2022 Mar 8;94(9):3800-3810. doi: 10.1021/acs.analchem.1c04402. Epub 2022 Feb 23.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry
  • Azides / chemistry
  • Catalysis
  • Click Chemistry* / methods
  • Copper / chemistry
  • Cycloaddition Reaction
  • Cysteine* / chemistry
  • Humans
  • Tandem Mass Spectrometry

Substances

  • Alkynes
  • Azides
  • Copper
  • Cysteine