Unbiased Mass Spectrometry Elucidation of the Targets and Mechanisms of Activity-Based Probes: A Case Study Involving Sulfonyl Fluorides

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.