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An Unbiased Proteomic Platform for ATE1-based Arginylation Profiling
Zongtao Lin1, Yixuan Xie1, Joanna Gongora1
1Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO 63110.
Biorxiv : the Preprint Server for Biology
|June 10, 2024
Summary
Protein arginylation, a crucial modification catalyzed by ATE1, is now identifiable using our novel activity-based arginylation profiling (ABAP) platform. This method accurately distinguishes arginylation from translation, revealing new modification sites.
Area of Science:
- Biochemistry
- Proteomics
- Post-translational Modifications
Background:
- Protein arginylation is a vital post-translational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1).
- Differentiating arginylation from translational arginine residues is challenging due to identical mass.
- Existing methods lack the specificity to unbiasedly identify arginylation substrates and sites.
Purpose of the Study:
- To develop a general activity-based arginylation profiling (ABAP) platform.
- To enable unbiased discovery of arginylation substrates and precise modification sites.
- To overcome the challenge of distinguishing arginylation from translational arginine residues.
Main Methods:
- Integration of isotopic arginine labeling into an ATE1 assay using biological lysates (ex vivo).
- Elimination of translational bias by avoiding live cells and ribosomal activity.
- Application of the ABAP platform to diverse sample types including peptides, proteins, cells, and tissues.
Main Results:
- Successfully identified 229 unique arginylation sites in human proteomes.
- Demonstrated the platform's applicability across various sample types with minimal input (20 μg).
- Validated representative arginylation sites and initiated functional follow-up studies.
Conclusions:
- The ABAP platform provides a robust method for bona fide arginylation identification.
- This technology facilitates the functional characterization of arginylation.
- The platform is globally applicable, paving the way for broader research into protein arginylation.
