Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of a “bottom up” proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement, yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted in the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodology might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1 but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.

中文翻译：

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16α-羟基雌酮：基于质谱的方法，用于鉴定与血液蛋白质形成的共价加合物。

雌酮代谢物 16α-羟基雌酮 (16αOHE1) 水平升高与多种疾病有关。作为一种亲电反应代谢物，与蛋白质的共价结合被认为是与 16αOHE1 相关的有害健康结果发生的可能机制之一。虽然基于质谱 (MS) 的方法目前被认为最适合监测蛋白质共价加合物的形成，但尚未提供这些方法用于鉴定 16αOHE1 共价加合物的应用。在本研究中，最终目标是确定最合适的方法来寻找 16αOHE1 衍生的共价加合物，体外16αOHE1。“自下而上”蛋白质组学方法的应用，包括对胰蛋白酶肽的 nanoLC-ESI-HRMS/MS 分析，允许鉴定 Hb 和 HSA 中 16αOHE1 加合物的多个位点。正如预期的那样，在通过还原或通过 Heyns 重排与 16αOHE1 形成的希夫碱稳定后，大多数加成肽发生在赖氨酸残基中，分别产生稳定的 α-羟胺和酮胺加合物。值得注意的是，丝氨酸残基也被鉴定为与 16αOHE1 共价修饰，据我们所知，这构成了酮亲电试剂作为基于羟基的亲核氨基酸目标的第一手报告。该ñ烷基 Edman 降解导致不适用于鉴定与 Hb 的 N 端缬氨酸形成的 16αOHE1 加合物，这很可能是由于所测试的衍生剂（异硫氰酸荧光素和异硫氰酸苯酯）在评估这些庞大的共价加合物时存在立体化学限制。尽管如此，将加成蛋白质消化为氨基酸导致检测到 16αOHE1 衍生的酮和 α-羟胺赖氨酸加合物。这种方法的简单性可能有利于临床研究，有可能通过制备这些加合物的合成标准来提供定量信息。