Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan.
Cell Rep Methods. 2022 Jan 14;2(1):100138. doi: 10.1016/j.crmeth.2021.100138. eCollection 2022 Jan 24.
Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7,000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.
基于激酶-底物关系鉴定细胞磷酸化途径是理解细胞生理功能调控的关键步骤。基于质谱的磷酸化蛋白质组学工作流程使得全面收集各种样本中单个磷酸化位点的信息成为可能。然而，目前仍然没有一种通用的方法可以根据激酶-底物关系在稀有细胞群体中揭示磷酸化网络。在这里，我们描述了一种基于基序的磷酸化蛋白质组学方法，结合了多重同位标记，其中激酶反应用于生成靶向磷酸肽，这些磷酸肽被添加到一个同位标记通道中以提高检测能力。概念验证实验证明，通过使用多个激酶对基序中心通道进行靶向磷酸肽的选择性和全面定量。从几十微克的起始材料中定量了超过 7000 个酪氨酸磷酸化位点。这种方法能够以基序为中心的方式对生理或病理调节下的多个磷酸化途径进行定量。
