Lin Zongtao, Xie Yixuan, Gongora Joanna, Liu Xingyu, Zahn Emily, Palai Bibhuti Bhusana, Ramirez Daniel H, Searfoss Richard M, Vitorino Francisca N, Karki Rashmi, Dann Geoffrey P, Zhao Chenfeng, Han Xian, MacTaggart Brittany, Lan Xin, Fu Dechen, Greenberg Lina, Zhang Yi, Lavine Kory J, Greenberg Michael J, Lv Dongwen, Kashina Anna, Garcia Benjamin A
Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, USA.
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.
Nat Chem Biol. 2025 Aug 25. doi: 10.1038/s41589-025-01996-z.
Protein arginylation is an essential post-translational modification catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating ribosomal bias and enabling bona fide arginylation identification. The method has been successfully applied to peptide, protein, cell, patient and mouse samples, with 235 unique arginylation sites revealed from human proteomes using 20 µg of input. Representative sites were validated and followed up for their biological functions. This global platform, applicable to various sample types, paves the way for functional studies of this difficult-to-characterize protein modification.
蛋白质精氨酰化是哺乳动物系统中由精氨酰 - tRNA - 蛋白质转移酶1（ATE1）催化的一种重要的翻译后修饰。精氨酰化的特点是将一个精氨酰基翻译后共价连接到蛋白质上，这使得它与具有相同质量的翻译精氨酸残基区分开来极具挑战性。在这里，我们提出了一个基于ATE1的通用精氨酰化分析平台，用于无偏倚地发现精氨酰化底物及其精确修饰位点。该方法将同位素精氨酸标记整合到利用生物裂解物（体外）而非活细胞的ATE1分析中，从而消除核糖体偏差并实现真正的精氨酰化鉴定。该方法已成功应用于肽、蛋白质、细胞、患者和小鼠样本，使用20μg输入物从人类蛋白质组中揭示了235个独特的精氨酰化位点。对代表性位点进行了验证并对其生物学功能进行了后续研究。这个适用于各种样本类型的全球平台为这种难以表征的蛋白质修饰的功能研究铺平了道路。
