Chang Alexis, Leutert Mario, Rodriguez-Mias Ricard A, Villén Judit
Department of Genome Sciences, University of Washington, Seattle WA 98195, USA.
bioRxiv. 2023 Jan 6:2023.01.05.522335. doi: 10.1101/2023.01.05.522335.
Phosphotyrosine (pY) enrichment is critical for expanding 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 handles 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.
通过基于质谱的蛋白质组学来扩展对细胞信号传导的基础和临床理解，磷酸酪氨酸（pY）富集至关重要。然而，由于昂贵的亲和试剂和手工操作，目前的pY富集方法每个样本成本高昂且重现性有限。我们提出了快速机器人磷酸酪氨酸蛋白质组学（R2-pY）方法，该方法使用磁性颗粒处理器和pY超结合剂或抗体。R2-pY可并行处理96个样本，从细胞裂解物到质谱进样只需2天，并能生成整体蛋白质组、磷酸蛋白质组和酪氨酸特异性磷酸蛋白质组样本。我们在用过氧钒酸盐和血清刺激的HeLa细胞上对该方法进行了基准测试，结果表明，输入1毫克肽可鉴定出4000多个独特的pY位点，重复样本间重现性良好，磷酸肽富集效率超过99%。R2-pY扩展了我们之前报道的R2-P2蛋白质组学和整体磷酸蛋白质组样本制备框架，为与整体蛋白质组和磷酸蛋白质组分析协同进行pY信号传导的大规模研究打开了大门。
