Life Sciences Institute, Department of Medical Oncology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Cancer Center, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Nat Commun. 2024 Jun 18;15(1):5221. doi: 10.1038/s41467-024-49517-1.
Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas. Here we develop an enrichable and multiple amino acids reactive Uaa, eFSY, and demonstrate that eFSY is MS cleavable when eFSY-Lys and eFSY-His are the cross-linking products. An identification software, AixUaa is developed to decipher eFSY mass cleavable data. We systematically identify direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa.
潜伏生物活性非天然氨基酸（Uaas）已广泛用于共价药物的开发和蛋白质相互作用物（如蛋白质、DNA、RNA 和碳水化合物）的鉴定。然而，由于蛋白质样品的复杂性和数据分析过程的复杂性，高通量鉴定 Uaa 交联产物具有挑战性。可富集的 Uaas 可以有效地降低蛋白质样品的复杂性并简化数据分析，但用这些 Uaas 从哺乳动物细胞样品中鉴定出的交联肽很少。在这里，我们开发了一种可富集和多反应性的 Uaa，eFSY，并证明当 eFSY-Lys 和 eFSY-His 是交联产物时，eFSY 是 MS 可裂解的。开发了一种鉴定软件 AixUaa 来破译 eFSY 质量可裂解的数据。我们系统地用 eFSY 和 AixUaa 鉴定了硫氧还蛋白 1（Trx1）和硒蛋白 M（SELM）的直接相互作用组。
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