Chemical and Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul 02792, Korea.
Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea.
Anal Chem. 2022 Mar 15;94(10):4192-4200. doi: 10.1021/acs.analchem.1c04188. Epub 2022 Mar 3.
Phosphorylation is a crucial component of cellular signaling cascades. It controls a variety of biological cellular functions, including cell growth and apoptosis. Owing to the low stoichiometry of phosphorylated proteins, the enrichment of phosphopeptides prior to LC-MS/MS is necessary for comprehensive phosphoproteome analysis, and quantitative phosphoproteomic workflows are typically limited by the amount of sample required. To address this issue, we developed an easy-to-establish, widely applicable, and reproducible strategy to increase phosphoproteomic signals from a small amount of sample without a phosphoenrichment step. By exploiting the multiplexing nature of isobaric labeling to generate a merged signal from multiple samples, and using a larger amount of enriched phosphopeptides as a carrier, we were able to increase trace amounts of phosphopeptides in the unpurified sample to an identifiable level and perform quantification using the reporter ion intensity of the isobaric tag. Our results showed that >1400 phosphopeptides were quantified from 250 ng of tryptic peptides prepared from cells. In a proof-of-concept of our strategy, we distinguished three types of lung cancer cell lines based on their quantitative phosphoproteomic data and identified changes in the phosphoproteome induced by drug treatment.
磷酸化是细胞信号级联反应的关键组成部分。它控制着多种生物细胞功能，包括细胞生长和细胞凋亡。由于磷酸化蛋白的低计量，在 LC-MS/MS 之前对磷酸肽进行富集对于全面的磷酸蛋白质组分析是必要的，而定量磷酸蛋白质组学工作流程通常受到所需样品量的限制。为了解决这个问题，我们开发了一种易于建立、广泛适用和可重复的策略，可在无需磷酸化富集步骤的情况下，从小量样本中增加磷酸蛋白质组学信号。通过利用同量异位标记的多路复用特性从多个样本中生成合并信号，并使用更多数量的富集磷酸肽作为载体，我们能够将未纯化样品中的痕量磷酸肽增加到可识别的水平，并使用同量异位标签的报告离子强度进行定量。我们的结果表明，从 250ng 酶解肽中可定量鉴定出 >1400 个磷酸肽。在我们策略的概念验证中，我们根据定量磷酸蛋白质组学数据区分了三种肺癌细胞系，并鉴定了药物处理诱导的磷酸蛋白质组变化。
