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

Abstract

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.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding

“…Given that ANGPTL4 binds to the same interface on free LPL and LPL•GPIHBP1 complexes, why does ANGPTL4 binding at 25°C trigger irreversible unfolding only in unbound LPL (and not in GPIHBP1-bound LPL)? A possible explanation rests in the observation that local fluctuations in protein conformations dominate at temperatures well below the melting temperature (T m ), whereas global fluctuations (unfolding/refolding) are predominant at temperatures closer to T m (39). We speculated that differences in T m of LPL and LPL•GPIHBP1 complexes could explain different susceptibilities to ANGPTL4-catalyzed LPL unfolding.…”

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

“…Another report 123 showed that by careful choice of what will be processed, namely, elimination of data that is unlikely to produce any processed results, overall analysis time can be sped up. It was shown that one can extract information from the isotope distribution pattern rather than from an entire uptake time course, 124 and a careful and systematic study 125 of the temperature-dependent factors in HDX MS experiments provided valuable examples and tools for studies of proteins over a wide temperature range.…”

HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data

“…We tested this approach on the unstructured peptide standard bradykinin and the intrinsically disordered aSyn. We hope that this new method, when used with scrutiny, will expand the types of differential HDX-MS experiments that can be done, as at present, it is rare to see comparisons between factors that affect the intrinsic rate (such as pH, temperature, ionic strength) 31,46 .…”