Abstract
HDX has emerged as a powerful tool to study protein conformation in native states. When HDX is analyzed at the peptide or protein level, only the overall deuterium uptake of peptides or proteins is obtained. Determination of deuterium incorporation at single amino acid resolution requires fragmentation which may induce “hydrogen scrambling”. ETD, as a nonergodic, fragmentation technique could yield a low level of hydrogen scrambling and therefore allow single residue localization of incorporated deuterium. A model peptide was used to evaluate this technique to obtain single residue resolution. Calcium binding protein, calmodulin, was studied with top-down and bottom-up approaches to compare the protein conformation for its apo- and holo- forms.Apo- and holo- forms of calmodulin were studied by top-down and bottom-up HDX-ETD experiments using a fully automatic HDX workflow station with Vanquish Binary pump N™, HESI source and a modified Thermo Scientific™ Orbitrap Ascend™ mass spectrometer. A full scan (MS) and an ETD MS2 scan were both conducted at the same time for bottom-up experiments. The MS full spectra were used to probe region of significant change in deuterium incorporation, and the ETD MS2 spectra were then used to pinpoint deuterium incorporation at the single amino acid level for the specific peptides that showed significant deuterium change.The synthetic peptide P1, HHHHHHIIKIIK, was prepared in both H2O and D2O at 10 µmol/L and kept at 4 ºC over 24 hours allowing complete deuterium exchange. The effect of source and capillary temperature on hydrogen scrambling and deuterium back exchange was measured, and MS parameters were tuned to limit scrambling. The low deuterium incorporation between c2 and c6 is indication of a low level of deuterium scrambling. The deuterium content for the c fragments of peptide P1 is comparable to what was reported in the literature.Top-down experiment of MS full scan with targeted ETD MS2 was performed for apo-, holo- calmodulin at different labeling time points, using optimized ETD reaction time of 5 msec. The deuterium incorporation of the intact level was measured. High sequence coverage was obtained from identified fragments from N and C terminals. HDExaminer was used to calculate the deuterium incorporation for each fragment and to generate the mirror plots. The plots clearly showed that there was more deuterium incorporation for the C-terminal than N-terminal. Based on the crystal structure, there were more conformation changes on C-terminal side than N-terminal side. Nearly single amino acid resolution for N- and C-terminal was obtained to improve the resolution. Bottom-up hydrogen/deuterium exchange with five labeling time points experiments were also conducted Consistent results were obtained from the two approaches. Overall, the apo- calmodulin had more deuterium incorporation than the holo- calmodulin form.