Global Profiling of Human Proteome Cysteine Residues via Lipoic Acid-Mediated Disulfide Exchange

Lipoic acid (LA), an endogenous small molecule, is widely utilized in metabolic regulation and disease intervention. While its mechanism of action has been largely attributed to protein lipoylation, the dynamic chemical properties of its five-membered cyclic disulfide moiety remain underexplored. Here, we designed and synthesized alkyne-functionalized lipoic acid-derived probes, LAN-yne and LAO-yne. Employing a chemoproteomic strategy, we systematically mapped the covalent targets of the LAN-yne across the cellular proteome and assessed their potential biological functions. Quantitative proteomic analysis identified 852 probe-modified proteins and 1,157 corresponding cysteine modification sites. This study demonstrates that LA’s endocyclic disulfide bond can be mimicked to mediate specific protein modification via thiol–disulfide exchange, providing novel molecular insights and a technical platform for investigating LA’s potential pharmacological mechanisms and structural derivatization efforts beyond lipoylation.

硫辛酸（Lipoic acid，LA）是一种内源性小分子，被广泛应用于代谢调控与疾病干预领域。尽管其作用机制长期以来被认为主要与蛋白质硫辛酰化修饰（protein lipoylation）相关，但该分子所含五元环二硫键基团的动态化学性质仍有待深入探究。本研究设计并合成了两种炔基修饰的硫辛酸衍生探针：LAN-yne与LAO-yne，采用化学蛋白质组学（chemoproteomics）策略系统绘制了LAN-yne在细胞蛋白质组中的共价结合靶点，并评估了这些靶点的潜在生物学功能。定量蛋白质组学分析共鉴定得到852个探针结合蛋白及1157个对应的半胱氨酸修饰位点。本研究证实，LA的环内二硫键可通过巯基-二硫键交换反应（thiol–disulfide exchange）介导特异性蛋白质修饰，为探索LA在硫辛酰化修饰之外的潜在药理机制及结构衍生化研究提供了全新的分子视角与技术平台。