Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes
Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes
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DOI：
10.1074/mcp.m116.058354
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发表时间：
2016-05-01
影响因子：
7
通讯作者：
Jaffe, Jacob D.
中科院分区：
文献类型：
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作者：
Abelin, Jennifer G.;Patel, Jinal;Jaffe, Jacob D.
分析翻译后修饰代表了表征细胞过程的基因表达数据的另一个维度。许多细胞对药物的反应是由细胞磷酸化信号的变化介导的。我们试图开发一个通用平台，在该平台上可以对数千个样品的磷酸信号应答进行分析，并创建了一种靶向MS测定法，该测定法对磷酸肽的减少代表性集进行分析，我们证明这些磷酸肽是对化学扰动剂的应答的强指标。我们研究了来源于用26种不同生物活性小分子处理的三种细胞系的样品中磷酸化位点的协调调节。通过聚类并从每个聚类中挑选1至2个代理成员，从这些发现研究中选择磷酸肽分析物。开发了一种定量的靶向平行反应监测测定法，以直接测量96个减少代表性的探针。用于蛋白水解消化、蛋白质定量、肽纯化和磷酸肽富集的样品处理已完全自动化，使得仅在3天内就可以同时处理96个样品，平板磷酸肽富集方差为12%。这种高度可重复的过程允许在类似于200个样品中检测到类似于95%的还原代表性磷酸肽探针，通过测量发现实验的处理条件下产生的新样品中的探针来评估该测定的性能，使用一小部分分析工作来概括更深入实验的观察结果。我们在新的实验中测量了这些探针，改变了治疗方法、细胞类型和时间点，以证明其普遍性。我们证明该检测方法对常见信号通路（例如MAPK、PI 3 K/mTOR和CDK）的破坏敏感。高通量、减少代表性的磷酸蛋白质组学检测提供了一个平台，用于比较一系列生物条件下的扰动，适用于分析数千个样品。我们相信，通过比较磷酸化蛋白质组学特征，该测定将证明对已知和新型药物和遗传机制的分类非常有用。
Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed similar to 95% of the reduced-representation phosphopeptide probes to be detected in similar to 200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and time-points to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.