Abstract
Hydrogen/deuterium exchange (H/D) was performed on ubiquitin, cytochrome c and myoglobin, and deuterium incorporation was investigated by electrospray ionization mass spectrometry (ESIMS) under several conditions, with different pH and different content of organic solvent. Circular dichroism (CD) was employed to observe conformational changes in proteins under the conditions for H/D exchange and ESIMS measurements. Comparing the results of H/D exchange experiments, ubiquitin was found to be the most stable among the three proteins against acid and organic solvent. Cytochrome c seemed to have interconvertible conformers under acidic condition with the addition of acetonitrile, though it was not indicated in CD spectra. Myoglobin appeared to be folded very loosely in mildly acidic condition and it was difficult to observe its H/D exchange rate by ESIMS, though its CD spectra showed little change under the conditions used for ESIMS analyses. It was possible to investigate slight conformational changes in proteins by utilizing H/D exchange and ESIMS analyses complementary to CD spectra.
