State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Lifeomics, National Center for Protein Sciences (Beijing), Beijing, China.
State Key Laboratory of NBC Protection for Civilian, Beijing, China.
Proteomics. 2024 Jan;24(1-2):e2300039. doi: 10.1002/pmic.202300039. Epub 2023 Aug 31.
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 ). AVP targeting peptides from human serum albumin (HSA) or plasma were captured by BTP-N 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.
有机磷化合物（OPs），如化学试剂和杀虫剂，由于其对不同氨基酸残基的不可逆膦酰化作用，形成不同的蛋白质加合物，对平民构成了严重威胁。然而，传统的分析方法在捕捉影响蛋白质功能的众多翻译后事件方面存在很大的局限性，特别是在鉴定低丰度 OP 加合物方面。本文首次采用炔基标记的 V 型试剂探针（AVP）和基于生物正交反应的叠氮化物富集连接子（BTP-N），开发了一种基于系统蛋白质组学的策略。AVP 靶向人血清白蛋白（HSA）或血浆中的肽段，通过 CuAAC 点击反应与 BTP-N 结合，用链霉亲和素珠富集，通过对苯甲酰酯键的选择性碱性水解释放，然后通过 LC-MS/MS 进行测序。该策略有助于在人血浆中的 163 种蛋白质上鉴定出 1115 个独特的 OP 加合物位点，涵盖了许多传统检测方法无法检测到的 OP 加合物。新型 OP 底物的全面覆盖为有毒 OP 的回顾性验证和/或剂量评估提供了一种通用且敏感的方法。
