High throughput discovery of functional protein modifications by Hotspot Thermal Profiling
High throughput discovery of functional protein modifications by Hotspot Thermal Profiling
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DOI：
10.1038/s41592-019-0499-3
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发表时间：
2019-09-01
期刊：
影响因子：
48
通讯作者：
Moellering, Raymond E.
中科院分区：
文献类型：
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作者：
Huang, Jun X.;Lee, Gihoon;Moellering, Raymond E.
质谱法能够对生物样品中翻译后修饰的蛋白质形态进行全局分析，但我们仍然缺乏系统预测甚至优先考虑哪些修饰位点可能干扰蛋白质功能的方法。在这里，我们描述了一种蛋白质组学方法，热点热分析，以检测位点特异性蛋白磷酸化对活细胞中数千种天然蛋白的热稳定性的影响。这种大规模平行的生物物理分析揭示了响应位点特异性磷酸化位点的整体蛋白质稳定性的变化，以及与蛋白质功能和结构相关的趋势。这种方法可以检测蛋白质结构的内在变化以及磷酸化引起的蛋白质-蛋白质和蛋白质-代谢物相互作用的外在变化。最后，我们证明了功能“热点”蛋白质修饰位点可以被发现并以高通量和公正的方式优先进行研究。这种方法适用于不同的生物体、细胞类型和翻译后修饰。
Mass spectrometry enables global analysis of posttranslationally modified proteoforms from biological samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method, Hotspot Thermal Profiling, to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect intrinsic changes to protein structure as well as extrinsic changes to protein-protein and protein-metabolite interactions resulting from phosphorylation. Finally, we show that functional 'hotspot' protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.