Voruganti Sudhakar, Kline Jake T, Balch Maurie J, Rogers Janet, Matts Robert L, Hartson Steven D
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, 74078, USA.
Bristol-Myers Squibb, Pennington, NJ, USA.
Methods Mol Biol. 2018;1709:139-162. doi: 10.1007/978-1-4939-7477-1_11.
Mass spectrometry assays demonstrate that Hsp90 inhibitors alter the expression of approximately one-quarter of the assayable proteome in mammalian cells. These changes are extraordinarily robust and reproducible, making "proteomics profiling" the gold standard for validating the effects of new Hsp90 inhibitors on cultured cells. Proteomics assays can also suggest novel hypotheses regarding drug mechanisms. To assist investigators in adopting this approach, this Chapter provides detailed protocols for conducting simple proteomics assays of Hsp90 inhibition. The protocols present a robust label-free approach that utilizes pre-fractionation of protein samples by SDS-PAGE, thereby providing reasonably good penetration into the proteome while addressing common issues with sample quality. The actual programming and operation of liquid chromatography-tandem mass spectrometers is not covered, but expectations for achievable performance are discussed, as are alternative approaches, common challenges, and software for data analysis.
质谱分析表明，Hsp90抑制剂可改变哺乳动物细胞中约四分之一可检测蛋白质组的表达。这些变化非常显著且可重复，使得“蛋白质组学分析”成为验证新型Hsp90抑制剂对培养细胞作用的金标准。蛋白质组学分析还可以提出有关药物作用机制的新假设。为帮助研究人员采用这种方法，本章提供了进行Hsp90抑制简单蛋白质组学分析的详细方案。这些方案提出了一种强大的无标记方法，该方法利用SDS-PAGE对蛋白质样品进行预分级分离，从而在解决样品质量常见问题的同时，对蛋白质组有相当好的覆盖度。本文不涉及液相色谱-串联质谱仪的实际编程和操作，但会讨论对可实现性能的期望，以及替代方法、常见挑战和数据分析软件。
