We’re thrilled to announce a collaboration between the renowned Jeff Keillor lab at the University of Ottawa and Dalriada’s protein mass spectrometry team. Recently published in Biochemistry, our research showcases the potential of substrate-free covalent binding kinetics by mass spectrometry (MS).
In the study, we focused on Transglutaminase 2 (TG2), an isozyme within the Transglutaminases (TGases) family, known for its multifaceted roles as both a transamidase and a G-protein. TG2, found ubiquitously in humans, is implicated in various disease pathologies including cancer, celiac disease and fibrosis. Traditionally, evaluation of TG2’s irreversible binding kinetics has involved biochemical assays with different substrates. However, our study uncovered a significant challenge: substrate binding events influenced TG2 conformational equilibria and overall activity, causing variations in the measured binding affinity of irreversible inhibitors based on substrate structure and concentration.
To overcome this challenge, Dalriada’s protein MS team utilized our mass spectrometry (MS)-based covalent kinetics platform powered by RapidFire-MS technology and our liquid handling system. This platform enables rapid and temporal sampling of enzyme and irreversible inhibitor reactions devoid of any reporter substrate. Our findings revealed that the remarkable increase in kinact/KI in the biochemical assay was primarily due to a decrease in KI. This aligned with the hypothesis that substrate binding and reaction lead to an activated enzyme form with a heightened affinity for irreversible inhibitors, while kinact remained unaffected by the substrate.
This work demonstrated that MS-based covalent kinetics assay not only can be applied to proteins that lack a biochemical assay but also offer advantages for those proteins with established biochemical assays.
Congratulations to the Jeff Keillor lab and Dalriada’s Dr. Taleb Sedighi, and Dr. Yasaman Heidari! 🎉