Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and m...

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Bibliographic Details
Published in:Journal of proteome research 2023-06, Vol.22 (6), p.1868-1880
Main Authors: Chang, Alexis, Leutert, Mario, Rodriguez-Mias, Ricard A., Villén, Judit
Format: Article
Language:English
Subjects:
HeLa Cells
Humans
Peptides - chemistry
Phosphopeptides - analysis
Phosphorylation
Phosphotyrosine - metabolism
Proteome - analysis
Proteomics - methods
Reproducibility of Results
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Summary:Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.
ISSN:1535-3893
1535-3907
DOI:10.1021/acs.jproteome.2c00850