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International audience; Trypanosomatid parasites of the genus Leishmania cause severe human diseases collectively termed leishmaniasis. Parasite ATP-binding proteins have emerged as potent targets for chemotherapeutic intervention. However, many parasite-specific ATP-binding proteins may escape current efforts in drug target identification, validation and deconvolution due to the lack of sequence conservation and functional annotation of these proteins in early branching eukaryotic trypanosomatids. Here, we selectively enriched for ATP-binding proteins from Leishmania donovani axenic promastigote and amastigote total protein extracts utilizing a Capture Compound™ (CC) linked to the ATP-competitive inhibitor staurosporine. As judged by in-gel kinase activity assay and competitive inhibition with free staurosporine, the CC specifically enriched for parasite phosphotransferases. Comparative nanoLC-MS(n) analysis identified 70 captured proteins, including 24 conserved protein kinases, and 32 hypothetical proteins with potential ATP-binding function. We identified conserved signature sequence motifs characteristic for staurosporine-binding protein kinases, and identified the hypothetical proteins LinJ.20.0280 and LinJ.09.1630 as novel ATP-binding proteins. Thus, functional enrichment procedures such as described here, combined with bio-informatics analyses and activity assays, provide powerful tools for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.BIOLOGICAL SIGNIFICANCE: Functional enrichment using a Capture Compound™ linked to the ATP-competitive inhibitor staurosporine provides a powerful new tool for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.