Neurotoxicity studies caused by exposure to acrylamide (AA) are of wide interest, but the methods for direct analysis of AA targets in living neuronal cells by cysteine profiling are still lacking. To address this, we developed a specific bioorthogonal probe, AAPA-P2, for chemical proteomics analysis of AA covalent binding sites. AAPA-P2 captured 754 target proteins, increasing the number of identified target proteins by 20-fold. Further screening revealed 96 proteins that are both highly sensitive and heavily modified by AAPA-P2, with validation performed on some potential key targets and binding sites. AA was found to induce neurotoxicity by binding to newly identified targets, Proteasome 26S Subunit, non ATPase 9 (PSMD9) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 (NDUFA5), interfering with the ubiquitin-proteasome system, and inducing mitochondria-dependent apoptosis. The present work provides an effective bioorthogonal probe tool for identifying covalent binding targets of acrylamide and offers new insights into the molecular mechanisms underlying acrylamide-induced neurotoxicity.

中文翻译：

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通过生物正交探针进行化学蛋白质组学分析，揭示小胶质细胞中丙烯酰胺的新靶点


暴露于丙烯酰胺 （AA） 引起的神经毒性研究引起了广泛关注，但仍然缺乏通过半胱氨酸分析直接分析活神经元细胞中 AA 靶标的方法。为了解决这个问题，我们开发了一种特异性生物正交探针 AAPA-P2，用于 AA 共价结合位点的化学蛋白质组学分析。AAPA-P2 捕获了 754 种靶蛋白，使鉴定出的靶蛋白数量增加了 20 倍。进一步筛选发现了 96 种蛋白质，这些蛋白质既高度敏感又受 AAPA-P2 严重修饰，并对一些潜在的关键靶点和结合位点进行了验证。发现 AA 通过与新发现的靶标蛋白酶体 26S 亚基、非 ATP 酶 9 （PSMD9） 和 NADH 脱氢酶（泛醌）1 α 亚复合物 5 （NDUFA5） 结合来诱导神经毒性，干扰泛素-蛋白酶体系统，并诱导线粒体依赖性细胞凋亡。这项工作为鉴定丙烯酰胺的共价结合靶点提供了一种有效的生物正交探针工具，并为丙烯酰胺诱导的神经毒性的分子机制提供了新的见解。