p-Nitrobenzyl mesylate (PNBM) is an alkylating agent used to identify direct kinase substrates [1]. The alkylation mechanism of p-Nitrobenzyl mesylate is to alkylate the phosphorothioate site to form a phosphorothioate epitope [2]. This epitope is detected by immunoblotting, immunoprecipitation, or immunoaffinity purification using a phosphorothioate-specific monoclonal antibody [3].
References:
[1] Haines E, Chen T, Kommajosyula N, et al. Palbociclib resistance confers dependence on an FGFR-MAP kinase-mTOR-driven pathway in KRAS-mutant non-small cell lung cancer[J]. Oncotarget, 2018, 9(60): 31572.
[2] Allen J J. Development and application of technologies to study individual kinase substrate relationships[D]. University of California, San Francisco, 2008.
[3] Shi W, Zhang G, Ma Z, et al. Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumour cell mitosis and impairs trastuzumab sensitivity to gastric cancer. Nat Commun. 2021; 12: 2812[J].
对硝基苄基甲磺酸酯（p-Nitrobenzyl mesylate; PNBM）是一种烷基化试剂，用于鉴定直接激酶底物[1]。p-Nitrobenzyl mesylate的烷化原理：烷基化硫代磷酸化位点，形成硫代磷酸酯表位[2]。用硫代磷酸酯特异性单克隆抗体通过免疫印迹、免疫沉淀或免疫亲和纯化来检测该表位 [3]。