biostudies-literatureSmith RJBill and Melinda Gates FoundationWellcome Trust102002https://www.ebi.ac.uk/biostudies/studies/S-EPMC9841287biostudies-literatureUnknown4(1)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).¹.STAR protocolsChemical pulldown combined with mass spectrometry to identify the molecular targets of antimalarials in cell-free lysates.PMC9841287218448/Z/19/Z105021/Z/14/Z203134/Z/16/ZDey GWyllie SLopez VCSyed AJMilne RPatterson SWiedemar NSmith RJfalseChemical pulldown combined with mass spectrometry to identify the molecular targets of antimalarials in cell-free lysates.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).¹.2023-01-01T00:00:00Z2023 Mar2026-04-08T12:29:05.748Z2025-02-19T03:27:33.456ZS-EPMC98412873660915310.1016/j.xpro.2022.102002