Wellcome Centre for Anti-infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Wellcome Centre for Anti-infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
STAR Protoc. 2023 Mar 17;4(1):102002. doi: 10.1016/j.xpro.2022.102002. Epub 2023 Jan 6.
Here, we provide a protocol using chemical pulldown combined with mass spectrometry (LC-MS/MS) to identify drug targets in Plasmodium falciparum. This approach works upon the principle that a resin-bound inhibitor selectively binds its molecular target(s) in cell-free lysates. We describe the preparation of drug beads and P. falciparum lysate, followed by chemical pulldown, sample fractionation, and LC-MS/MS analysis. We then detail how to identify specifically bound proteins by comparing protein enrichment in DMSO-treated relative to drug-treated lysates via quantitative proteomics. For complete details on the use and execution of this protocol, please refer to Milne et al. (2022)..
在这里，我们提供了一个使用化学拉下结合质谱（LC-MS/MS）来鉴定恶性疟原虫药物靶点的方案。该方法基于树脂结合的抑制剂在无细胞裂解物中选择性结合其分子靶标（s）的原理。我们描述了药物珠和恶性疟原虫裂解物的制备，然后进行化学拉下、样品分级和 LC-MS/MS 分析。然后，我们详细介绍了如何通过比较 DMSO 处理与药物处理裂解物中蛋白质的丰度差异，通过定量蛋白质组学来鉴定特异性结合的蛋白质。有关该方案使用和执行的完整详细信息，请参阅 Milne 等人（2022 年）。
