Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, Toronto, Ontario, Canada.
Nat Biotechnol. 2011 Jun 26;29(7):653-8. doi: 10.1038/nbt.1905.
Signaling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors. However, we have little quantitative data regarding the kinetics with which such networks assemble and dissolve to generate specific cellular responses. To address this deficiency, we designed a mass spectrometry method, affinity purification-selected reaction monitoring (AP-SRM), which we used to comprehensively and quantitatively investigate changes in protein interactions with GRB2, an adaptor protein that participates in a remarkably diverse set of protein complexes involved in multiple aspects of cellular function. Our data reliably define context-specific and time-dependent networks that form around GRB2 after stimulation, and reveal core and growth factor-selective complexes comprising 90 proteins identified as interacting with GRB2 in HEK293T cells. Capturing a key hub protein and dissecting its interactions by SRM should be equally applicable to quantifying signaling dynamics for a range of hubs in protein interaction networks.
信号通路通常通过可诱导的蛋白质-蛋白质相互作用进行组织，这些相互作用由衔接蛋白介导，将激活的受体与细胞质效应物连接起来。然而，我们对于这些网络组装和分解以产生特定细胞反应的动力学知之甚少。为了解决这一不足，我们设计了一种质谱方法，即亲和纯化-选择反应监测（AP-SRM），我们用它来全面、定量地研究与衔接蛋白 GRB2 相互作用的蛋白质的变化，GRB2 参与了一组非常多样化的蛋白质复合物，涉及细胞功能的多个方面。我们的数据可靠地定义了刺激后围绕 GRB2 形成的特定于上下文和随时间变化的网络，并揭示了核心和生长因子选择性复合物，其中包含 90 种被鉴定为与 HEK293T 细胞中的 GRB2 相互作用的蛋白质。通过 SRM 捕获关键枢纽蛋白并剖析其相互作用，应该同样适用于量化蛋白质相互作用网络中一系列枢纽的信号转导动态。
