RESUMO
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal exchange reporters (IERs), which directly monitor changes to exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical exchange correction, thereby improving reproducibility.
