Comprehensive insight on protein modification by V‐type agent: A chemical proteomic approach employing bioorthogonal reaction

Abstract Organophosphorus compounds (OPs) such as chemical agents and pesticides are posing critical threats to civilians due to their irreversible phosphonylation of diverse amino acids residues forming different protein adducts. However, traditional analytical approaches are quite limited in capturing the myriad of post‐translational events that affect protein functions, especially in identifying the low‐abundance OP adducts. Herein a systematic proteomic strategy based on a typical click‐enrich‐release‐identify bioorthogonal operation was firstly developed by employing an alkynyl‐tagged V‐type agent probe (AVP) and a biotin‐based azido‐enrichment linker (BTP‐N 3 ). AVP targeting peptides from human serum albumin (HSA) or plasma were captured by BTP‐N 3 via CuAAC click reaction, enriched by streptavidin beads, released by selective alkaline hydrolysis of phenacyl ester bond, and subsequently sequenced by LC‐MS/MS. This strategy has helped identifying 1115 unique OP adduction sites on 163 proteins in human plasma, and covers lots of OP adducts that cannot be achieved by traditional detection methods. The comprehensive coverage of novel OP substrates provided a general and sensitive approach to retrospective verification and/or dose assessment of toxic OPs.