Abstract
During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.