Schmidt Christian, Breyer Felicitas, Blum Marc-Michael, Thiermann Horst, Worek Franz, John Harald
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.
Anal Bioanal Chem. 2014 Aug;406(21):5171-85. doi: 10.1007/s00216-014-7706-y. Epub 2014 Mar 21.
Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and μ-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (μHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the ε-amino group of a formerly phosphonylated lysine and the side chain of an adjacent acidic glutamic acid residue.
已知有毒有机磷化合物（如农药和神经毒剂）会与不同氨基酸的亲核侧链发生反应（磷酸化），从而与内源性蛋白质形成加合物。最常见的是，与丝氨酸、酪氨酸或苏氨酸残基的结合被描述为与毒理学效应（如乙酰胆碱酯酶和神经病变靶酯酶）相关，或作为暴露后分析的生物标志物（如白蛋白和丁酰胆碱酯酶的验证）。因此，鉴定新的蛋白质靶点可能有助于更好地理解这些化合物的毒理学，揭示新的生物分析验证工具，并增进对化学反应性的了解。在本研究中，我们在体外研究了泛素（Ub）与V型神经毒剂中国VX、俄罗斯VX和VX的反应。Ub是一种普遍存在的蛋白质，分子量为8564.8Da，存在于细胞外和细胞内空间，在几个重要过程（如蛋白酶体降解、DNA修复、蛋白质周转和内吞作用）中发挥重要的生理作用。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和与紫外检测及电喷雾电离质谱在线联用的μ高效液相色谱（μHPLC-UV/ESI MS）对反应产物进行了分析。我们的结果首次证明生成了至少单、二和三膦酰化Ub加合物的复杂混合物。令人惊讶的是，肽质量指纹分析结合MALDI和ESI MS/MS显示，膦酰化在Ub生物学功能所必需的7个表面暴露赖氨酸残基中的至少6个上具有高度选择性。发现这些反应产物不会老化。此外，我们在此首次报告，膦酰化通过在先前膦酰化赖氨酸的ε-氨基与相邻酸性谷氨酸残基的侧链之间形成异肽键诱导分子内环化。
