Restraining Expansion of the Peak Envelope in H/D Exchange-MS and Its Application in Detecting Perturbations of Protein Structure/Dynamics

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Abstract
Hydrogen/deuterium exchange (H/DX) mass spectrometry (MS) is increasingly applied to problems in protein structural biology in order to map protein dynamics and identify sites of interactions. In theory, an MS-based readout of deuterium label incorporation can overcome the concentration, size, purity, and complexity limitations inherent in NMR-based measurements of exchange; however, in practice, these advantages are reduced due to spectral interference and dilution of the sample in deuterium oxide (D2O). In this study, we demonstrate that popular H/DX labeling strategies aggravate the interference problem and that significant recovery of spectral capacity may be achieved with a “minimalist” strategy. Simulations of peptide deuteration justify large reductions in the level of D2O used in labeling experiments, as well as reduced numbers of peaks used in making relative labeling measurements between biochemical states of a protein. To demonstrate the utility of a minimalist approach, calmodulin was interrogated in a bottom-up H/DX-MS workflow, and sensitivity to the addition of Ca2+ as a structural perturbation was measured as a function of % D2O and the number of peaks used in quantitating deuteration level. It is shown that high sensitivity to change is preserved with deuteration levels of 5.0 ± 1.1 (apo-CaM) and 1.4 ± 1.3% (holo-CaM) using 10% D2O in the labeling experiment. Further, only two peaks of a peptide peak distribution are needed to sensitively monitor changes in protein structure, dynamics, or both.