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<p>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.</p><p></p>

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An unbiased proteomic platform for ATE1-based arginylation profiling

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年8月25日

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  • Zongtao Lin,
  • Yixuan Xie,
  • Joanna Gongora,
  • Xingyu Liu,
  • Emily Zahn,
  • Bibhuti Bhusana Palai,
  • Daniel H. Ramirez,
  • Richard M. Searfoss,
  • Francisca N. Vitorino,
  • Rashmi Karki,
  • Geoffrey P. Dann,
  • Chenfeng Zhao,
  • Xian Han,
  • Brittany MacTaggart,
  • Xin Lan,
  • Dechen Fu,
  • Lina Greenberg,
  • Yi Zhang,
  • Kory J. Lavine,
  • Michael J. Greenberg,
  • Dongwen Lv,
  • Anna Kashina,
  • Benjamin A. Garcia

摘要

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.