Journal
ANALYTICAL CHEMISTRY
Volume 93, Issue 35, Pages 11946-11955Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c01591
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Funding
- National Science Foundation Graduate Research Fellowship [2018255830]
- National Institutes of Health [DA043571, GM037537]
- Robbins Family-MRA Young Investigator Award from the Melanoma Research Alliance
- University of Virginia Cancer Center (NCI Cancer Center) [5P30CA044579-27]
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Chemical proteomics is a widely used method for investigating protein activity and small molecule ligand binding. LC-MS/MS is employed to assess covalent probe binding and inhibition, providing molecular information on targeted proteins and probe-modified sites. Key bioanalytical conditions have been revealed to guide future direct target site identification of complex irreversible probes and inhibitors.
Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probemodified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.
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