Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely used chemically cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using the iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, we evaluated the key sample preparation steps. We also performed a mass tolerant database search strategy using MSFragger software. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.