Unbiased Mass Spectrometry Elucidation of the Targets and Mechanisms of Activity-Based Probes: A Case Study Involving Sulfonyl Fluorides
2018-09-07T00:00:00Z (GMT) by
The elucidation of protein/drug interactions remains a major challenge in drug discovery. Liquid chromatography–tandem mass spectrometry has emerged as a tremendously powerful technology for this endeavor, but its full potential has yet to be realized owing in part to unresolved challenges in data analysis. Herein, we demonstrate how tandem mass spectrometry can comprehensively map small molecule/peptide adducts when combined with unconstrained sequencing. Using a published sulfonyl fluoride activity-based probe as a model system, this method enabled the discovery of several unreported sites of interaction with its target proteins. Crucially, this probe was found to undergo quantitative displacement and hydrolysis from the target protein’s active site. Isotopic labeling experiments provided a mechanistic rationale for the observed hydrolysis that involves neighboring-group participation. A chemical biology tagging strategy that leverages the probe’s observed lability was developed and shown to be compatible with the original small molecule inhibitor in discovery profiling experiments.