Protein hydrogen exchange determined by mass spectrometry: A useful tool for studying the kinetics and thermodynamics of protein unfolding

Protein amide hydrogen exchange has long been used as a sensitive probe of protein high-order structure and structural changes. The principal method of determining rates of hydrogen exchange in proteins has been multi-dimensional NMR. However, NMR is limited to the study of small, highly soluble proteins. A new method, based on using different hydrogen/deuterium exchange techniques, protein fragmentation and mass spectrometry to determine dynamics of protein unfolding, was developed. In this method, the protein can be fragmented by an acid protease after the protein undergoes H/D exchange. Since the protein is digested into small peptides before the analysis, there is no limit in the size of the protein that can be studied. Different hydrogen/deuterium exchange labeling techniques in this new method were first evaluated using rabbit muscle aldolase (Mr 157 kDa) as a model. The method was then used to study the unfolding process of aldolase in a denaturant, urea. The hydrogen exchange results show that three domains of aldolase unfolded cooperatively with different rate constants in urea. Although the unfolding domains do not correlate well with units of secondary structure, the unfolding rates do con-elate with exposure of the amide hydrogens to the solvent. Kinetic fitting for the data of a time course study of aldolase unfolding suggested that the aldolase unfolded sequentially in urea. This new method can be applied to determine the stability of the partly unfolded states under physiological conditions by extrapolating results determined under denaturing conditions. When collision induced dissociation MS/MS was used with this method, more detailed structural information could be determined. Combining hydrogen/deuterium exchange, protein fragmentation, and mass spectrometry provides a powerful tool in the study of protein structural changes