Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

Abstract

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 2 H-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 2 H during separation based on their disulfidedependent 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.

Orders-of-Magnitude Larger Force Demonstrated for Dielectrophoresis of Proteins Enabling High-Resolution Separations Based on New Mechanisms

“…These proteins were chosen because these specific proteins were known to be well behaved and generated reproducible behaviors in an electric field across any number of platforms . − Each protein began to collect at an applied potential unique to that protein. At higher values of applied potential, the concentration within the collection area increased immediately and the intensity increased linearly with time of voltage applied.…”

“…In the present work, a set of three proteins were tested on a gradient insulator-based dielectrophoresis device (g-iDEP) under DC conditions (Figure ). The specific proteins were chosen because their electrophoretic behaviors have been recorded and this aspect of the data could be quantified and compared to literature examples. − The proteins exhibited behaviors consistent with negative dielectrophoresis, and each protein gave a unique electrokinetic mobility ratio (EKMr) according to distinguished onset potential for capturing. For each protein, the concentrated bolus profile allowed for quantification of the large dielectrophoretic forces present and these were observed to be reasonably consistent with previous electrophoretic results and theoretical calculations.…”

Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method

“…HDX‐MS has become a useful tool for the analysis of protein conformation, which enables mass analysis at intact protein levels to explore global changes in protein structure and stability . HDX occurs primarily on the backbone amide hydrogens of a protein because they are distributed along the polypeptide backbone and form a basic hydrogen bond network of secondary structures …”

“…HDX-MS has become a useful tool for the analysis of protein conformation, which enables mass analysis at intact protein levels to explore global changes in protein structure and stability. [44][45][46][47] HDX occurs primarily on the backbone amide hydrogens of a protein because they are distributed along the polypeptide backbone and form a basic hydrogen bond network of secondary structures. 48,49 In this work, HDX-MS was carried out to probe the binding effect The MD results indicated that the binding sites of the four IAs were located in the pocket around heme in Mb, and the order of the binding free energy values calculated using MD was consistent with F I G U R E 7 HDX-MS spectra of Mb and complexes of Mb with IAs.…”

Controlling H3+ Formation From Ethane Using Shaped Ultrafast Laser Pulses

“…The intramolecular migration of hydrogen continues to be an active area of investigation in ultrafast science [1][2][3][4][5][6][7][8][9][10][11] with implications for topics ranging from combustion [12] to peptide dissociation [13] and characterizing conformational differences in molecules [14,15]. In some cases the migration of hydrogen leads to the formation of new molecular ions, such as H + 3 [5,[16][17][18][19][20][21], by processes such as H 2 roaming or double hydrogen migration [18,19,22,23].…”