近日，美国芝加哥大学Raymond E. Moellering及其研究团队利用热点热分析方法实现对功能性蛋白的高空量挖掘。相关论文2019年9月发表在国际学术期刊《自然—方法学》上。

研究人员开发了一个名为热点热分析（Hotspot Thermal Profiling）的蛋白质组学方法，可以检测位点特异性蛋白质磷酸化对活细胞中数千种天然蛋白质的热稳定性的影响。这种大规模平行的生物物理分析揭示了响应于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。该方法可以检测蛋白质结构的内在变化以及由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，研究人员发现，通过这个方法，功能性“热点”蛋白质修饰位点能够被鉴定出来，并以高通量和无偏倚的方式优先进行研究。该方法适用于不同的生物、细胞类型和翻译后修饰。

据悉，质谱能够对来自生物样品的翻译后修饰的蛋白质进行全局分析，但科学家仍然缺乏能够系统性预测或优先考虑哪些修饰位点可能扰乱蛋白质功能的方法。

附：英文原文

Title: High throughput discovery of functional protein modifications by Hotspot Thermal Profiling

Author: Jun X. Huang, Gihoon Lee, Kate E. Cavanaugh, Jae W. Chang, Margaret L. Gardel, Raymond E. Moellering

Issue&Volume: Volume 16 Issue 9

Abstract: 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.

DOI: 10.1038/s41592-019-0499-3

Source:https://www.nature.com/articles/s41592-019-0499-3