Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.
J Proteome Res. 2023 Jun 2;22(6):1868-1880. doi: 10.1021/acs.jproteome.2c00850. Epub 2023 Apr 25.
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.
磷酸化酪氨酸（pY）富集对于通过基于质谱的蛋白质组学扩大对细胞信号转导的基础和临床理解至关重要。然而，由于昂贵的亲和试剂和手动处理，当前的 pY 富集方法每样本成本高且重现性有限。我们提出了快速机器人磷酸化蛋白质组学（R2-pY），它使用磁性颗粒处理器和 pY 超级结合剂或抗体。R2-pY 可以同时处理多达 96 个样本，从细胞裂解物到质谱注射需要 2 天，并且可以得到全局蛋白质组学、磷酸化蛋白质组学和酪氨酸特异性磷酸化蛋白质组学样本。我们在过钒酸盐和血清刺激的 HeLa 细胞上对该方法进行了基准测试，报告了来自 1 毫克肽输入的超过 4000 个独特的 pY 位点，重复之间具有很强的重现性，磷酸肽富集效率超过 99%。R2-pY 扩展了我们之前报道的 R2-P2 蛋白质组学和全局磷酸化蛋白质组学样品制备框架，为大规模研究 pY 信号与全局蛋白质组和磷酸化蛋白质组谱的协同作用打开了大门。
