In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography–mass spectrometry (LC–MS). A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.儿茶酚雌激素（Catechol estrogens, CEs）是一类代谢性亲电试剂，可与细胞蛋白质发生活跃的共价相互作用，进而影响分子功能。目前尚无可行方法可在活体系统中鉴定其结合蛋白。本研究开发了一种基于点击化学的分析方法：以炔雌醇（Ethinylestradiol, EE2）作为前驱体探针，结合定量蛋白质组学技术，实现CEs蛋白质靶点的鉴定，并对其结合强度进行分类。通过在肝微粒体中进行原位代谢转化与点击反应，探针捕获CEs-蛋白质复合物，经胰蛋白酶消化、还原胺化法进行稳定同位素标记后，采用液相色谱-质谱联用法（Liquid Chromatography–Mass Spectrometry, LC-MS）进行分析。最终共重复鉴定得到334种肝脏蛋白质（n≥2）；基于前体质量映射，其中274种鉴定蛋白被归类为强结合蛋白。结合强度进一步通过氘氢比（活性探针/溶剂）进行量化：259种强结合蛋白的D/H＞5.25；46种弱结合蛋白的5.25＞D/H＞1；另有5种非特异性结合蛋白（角蛋白）的D/H＜1。本研究通过添加共价对照（强结合物）与非共价对照（弱结合物），以及对细胞色素c进行体外验证（其D/H=5.9），证实了上述结果——细胞色素c可与CEs发生共价结合。众多鉴定得到的强结合蛋白，如谷胱甘肽转移酶、儿茶酚-O-甲基转移酶、超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶及细胞色素c等，均参与细胞氧化还原过程或解毒活动。研究还发现，CEs结合可抑制细胞色素c的超氧化物氧化酶活性，提示CEs修饰可能改变细胞蛋白质的氧化还原功能。由于结构相似性与惰性炔基的存在，EE2探针大概率可广谱捕获CEs的蛋白质靶点。因此，本策略可用于探索CEs修饰在活体系统中的生物学影响。