Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics

作者：Alexis Chang, Mario Leutert, Ricard A. Rodríguez‐Mias, Judit Villén · 发表于：Journal of Proteome Research · 年份：2023 · DOI：10.1021/acs.jproteome.2c00850 · 被引用次数：21 · 研究领域：Advanced Proteomics Techniques and Applications、Mass Spectrometry Techniques and Applications、Advanced Biosensing Techniques and Applications

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.