| Autor: |
McCloud RL; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Yuan K; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Mahoney KE; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Bai DL; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Shabanowitz J; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Ross MM; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Hunt DF; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States., Hsu KL; Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States.; Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, United States.; University of Virginia Cancer Center, University of Virginia, Charlottesville, Virginia 22903, United States. |
| Abstrakt: |
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 probe-modified 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. |
