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Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States
- Shen, Yue, Zhao, Xiuxiu, Wang, Guanbo, Chen, David D. Y.
- Analytical chemistry 2019 v.91 no.6 pp. 3805-3809
- capillary electrophoresis, deuterium, gases, heme, isomers, isotope labeling, lysozyme, mass spectrometry, molecular weight, stable isotopes
- Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted ²H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with ²H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.