Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling
Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling
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DOI：
10.1074/mcp.o115.054940
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发表时间：
2016-02-01
影响因子：
7
通讯作者：
Paulovich, Amanda G.
中科院分区：
文献类型：
--
作者：
Kennedy, Jacob J.;Yan, Ping;Paulovich, Amanda G.
细胞信号转导研究的一个主要目标是量化扰动后的磷酸化药效学。研究细胞磷酸化信号的传统方法一次测量一个分析物，标准化程度很低，使它们不足以询问网络生物学，并导致临床前研究的不可重复性。在这项研究中，我们测试了通过将基于固定化金属亲和层析(IMAC)的磷酸肽富集物与靶向、多反应监测(MRM)质谱仪相结合来规避这些问题的可行性，以实现对磷酸化信号响应的精确、特异、标准化、多重量化。建立了一种针对DNA损伤的磷分析物的多重固定化金属亲和层析多反应监测方法，并对其进行了分析表征和部署，以生成经历遗传毒性应激的原代和永生化的人类细胞的磷-药效曲线。多重分析的线性范围为~gt；=3个数量级，柱上定量下限的中位数为0.64 fmol，批内变异中值为9.3%，批间变异中值为12.7%，总变异系数的中位数为16.0%。多重固定化金属亲和层析-多反应监测分析方法能够可靠地定量检测DNA损伤后人类细胞中的107个DNA损伤反应亚磷酸盐。这些化验结果已作为社区资源公开提供。该方法普遍适用，允许对信令网络进行广泛的询问。
A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatographymultiple reaction monitoring assay targeting phosphoanalytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of >= 3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intraassay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography-multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.