An unbiased proteomic platform for ATE1-based arginylation profiling
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.
Authors
- Francisca N. de Luna Vitorino (ORCID: https://orcid.org/0000-0001-8543-2299)
- Kory J. Lavine (ORCID: https://orcid.org/0000-0003-1948-9945)
- Brittany MacTaggart (ORCID: https://orcid.org/0000-0001-7674-6042)
- Rashmi Karki (ORCID: https://orcid.org/0000-0002-8494-517X)
- Yi Zhang (ORCID: https://orcid.org/0000-0001-8170-9983)
- Lina Greenberg (ORCID: https://orcid.org/0000-0002-6066-3135)
- Richard Searfoss (ORCID: https://orcid.org/0000-0001-6235-2595)
- Daniel H. Ramirez (ORCID: https://orcid.org/0000-0003-3288-9951)
- Benjamin A. Garcia (ORCID: https://orcid.org/0000-0003-3596-4750)
- Geoffrey P. Dann
- Anna Kashina (ORCID: https://orcid.org/0000-0002-0243-6866)
- Zongtao Lin (ORCID: https://orcid.org/0000-0002-6017-338X)
- Xian Han (ORCID: https://orcid.org/0000-0002-3802-6420)
- Michael J. Greenberg (ORCID: https://orcid.org/0000-0003-1320-3547)
- Dongwen Lv (ORCID: https://orcid.org/0000-0003-4677-8996)
- Yixuan Xie (ORCID: https://orcid.org/0000-0002-8512-6053)
- Emily Zahn
- Bibhuti Bhusana Palai
- Xingyu Liu (ORCID: https://orcid.org/0009-0003-3815-4953)
- Chenfeng Zhao (ORCID: https://orcid.org/0000-0001-9952-0628)
- Xin Lan (ORCID: https://orcid.org/0009-0009-1615-2846)
- Dechen Fu
- Joanna Gongora
Institutions
- The University of Texas at San Antonio Health Science Center (US)
- Washington University in St. Louis (US)
- Augusta University (US)
- Saint Louis Zoo (US)
- Case Western Reserve University (US)
- University of Pennsylvania (US)
Publication Details
- Journal
- Nature Chemical Biology
- Published
- 2025-08-25
- DOI
- https://doi.org/10.1038/s41589-025-01996-z
- Citations
- 4
- Primary Topic
- Ubiquitin and proteasome pathways
- Type
- article
- Field-Weighted Citation Impact
- 3.22
Funders
- National Science Foundation
- American Heart Association
- Children's Discovery Institute
- National Institutes of Health
- University of Texas Health Science Center at San Antonio
- Washington University School of Medicine in St. Louis
- National Institute on Aging
- National Heart, Lung, and Blood Institute
- National Cancer Institute
- National Institute of General Medical Sciences
- National Institute of Diabetes and Digestive and Kidney Diseases
- National Institute of Neurological Disorders and Stroke
- Division of Chemistry
- Eunice Kennedy Shriver National Institute of Child Health and Human Development