Department of Chemistry , Duke University , Durham , North Carolina 27708 , United States.
J Am Soc Mass Spectrom. 2020 Feb 5;31(2):217-226. doi: 10.1021/jasms.9b00041. Epub 2019 Nov 22.
Recently, several mass-spectrometry- and protein-denaturation-based proteomic methods have been developed to facilitate protein target discovery efforts in drug mode-of-action studies. These methods, which include the stability of proteins from rates of oxidation (SPROX), pulse proteolysis (PP), chemical denaturation and protein precipitation (CPP), and thermal proteome profiling (TPP) techniques, have been used in an increasing number of applications in recent years. However, while the advantages and disadvantages to using these different techniques have been reviewed, the analytical characteristics of these methods have not been directly compared. Reported here is such a direct comparison using the well-studied immunosuppressive drug, cyclosporine A (CsA), and the proteins in a yeast cell lysate. Also described is a one-pot strategy that can be utilized with each technique to streamline data acquisition and analysis. We find that there are benefits to utilizing all four strategies for protein target discovery including increased proteomic coverage and reduced false positive rates that approach 0%. Moreover, the one-pot strategy described here makes such an experiment feasible, because of the 10-fold reduction in reagent costs and instrument time it affords.
最近，已经开发了几种基于质谱和蛋白质变性的蛋白质组学方法，以促进药物作用机制研究中的蛋白质靶标发现工作。这些方法包括蛋白质氧化速率稳定性（SPROX）、脉冲蛋白水解（PP）、化学变性和蛋白质沉淀（CPP）以及热蛋白质组学分析（TPP）技术，近年来在越来越多的应用中得到了使用。然而，尽管已经对使用这些不同技术的优缺点进行了综述，但这些方法的分析特性尚未进行直接比较。本研究使用经过充分研究的免疫抑制剂环孢素 A（CsA）和酵母细胞裂解物中的蛋白质进行了这种直接比较。还描述了一种可以与每种技术一起使用的一锅策略，以简化数据采集和分析。我们发现，利用这四种策略进行蛋白质靶标发现有很多好处，包括增加蛋白质组覆盖范围和降低接近 0%的假阳性率。此外，由于这种实验可以节省试剂成本和仪器时间，因此这里描述的一锅策略使其变得可行。
