Analysis of Temperature-Dependent H/D Exchange Mass Spectrometry Experiments

H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX–MS, proteins are incubated in D₂O as a function of time at constant temperature (T). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T-dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX–MS data in the context of T-dependent protein dynamics. Specifically, it has not been possible thus far to separate T-induced changes of the chemical labeling step (k_ch) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T-dependent HDX–MS profiles into contributions from k_ch(T), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential k_ch(T) dependence. Just below the melting temperature (T_m) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T, while global events become prevalent closer to T_m. About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive ΔH and ΔS. Many of the remaining sites had negative ΔH and ΔS, thereby accounting for the shallowness of the experimental HDX–MS profiles at low T. In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T-dependent high-throughput screening applications by HDX–MS.