Direct Target Site Identification of a Sulfonyl–Triazole Covalent Kinase Probe by LC-MS Chemical Proteomics

Abstract

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.

Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation

“…47,48 In addition, fragmentation of sulfonyl-triazole probes has been harnessed for site-of-labeling studies. 49 When combined with custom isobaric data analysis algorithms, these modifications should also provide an avenue to improve MS2-level quantification of peptide labeling. We anticipate that realizing the full potential of labile search algorithms in chemoproteomic applications may depend on advances in these algorithms to take full advantage of partial fragmentation, such as that observed for the CBCC reagents described here.…”

“…First, we present the low-cost synthesis of an isotopically labeled pair of biotin-azide reagents, which compares favorably to the cost and complexity of established isotopically labeled reagents, both azide-containing and those that feature cysteine-reactive electrophiles. ,,, Our demonstration of the labile ion search features built into FragPipe should also provide a generalizable computational platform for others interested in leveraging fragmentation of chemoproteomics samples. Exemplifying the utility of such studies, gas-phase fragmentation of cysteines modified by covalent drugs, such as ibrutinib, has been leveraged to improve the identification of labeled cysteine residues. , In addition, fragmentation of sulfonyl-triazole probes has been harnessed for site-of-labeling studies . When combined with custom isobaric data analysis algorithms, these modifications should also provide an avenue to improve MS2-level quantification of peptide labeling.…”

Reactivity-based chemical-genetic study of protein kinases

“…More recently, a sulfonyl-triazole analog of XO44 (KY-26) was designed to modify both lysine and tyrosine residues on protein kinases in another chemoproteomic analysis. 85 This shows that irreversible inhibitors are well-suited not only for chemical genetics but they also serve as ideal scaffolds for the development of chemical probes for use in other applications such as activity-based protein profiling (ABPP) or high-throughput screens (HTS).…”

Mapping the Protein Kinome: Current Strategy and Future Direction

“…After capturing the labeled proteins, up to 133 endogenous kinases have been identified [ 82 ]. KY-26, which has been modified based on XO-44, performs better in kinome identification [ 83 ]. Although probes have been developed to cover most kinases, it is still necessary to develop specific probes for hard-to-detect kinases.…”