Protein digestion using a cysteine-specific backbone cleavage reagent.

Mass spectrometry (MS)-based protein analysis is an indispensable tool in modern biomedical research. A key step in sample preparation is proteolytic digestion using enzymes with well-defined amino acid specificity, such as trypsin, chymotrypsin, and StaphV8 protease, which cleave at basic, aromatic, and acidic residues, respectively. The absence of cysteine (Cys)-specific cleavage methods is a gap in the current protein analysis toolbox. Herein, we report a chemical reagent (1) that selectively cleaves the N-terminal amide bond of Cys residues in proteins. Using glutathione as a model peptide, we investigated the reaction kinetics in detail and identified optimized conditions for protein cleavage. Using thioesterase as a model protein, we further demonstrated that 1 is fully compatible with modern MS-based proteomics workflows, including in-gel digestion, where it can be used in combination with existing proteases. This reaction proceeds rapidly and selectively in aqueous buffers, affording high yields while converting the reactive Cys side-chain thiol into a chemically inert five-membered heterocyclic moiety. This transformation eliminates the need for the commonly employed iodoacetamide capping step and introduces a distinct mass tag that facilitates downstream data analysis. Overall, these features establish 1 as a robust and practical new tool for protein analysis.