Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase.

The protein fragmentation/mass spectrometry method described by Zhang and Smith [(1993) Protein Sci. 2, 522-531] has been extended to measure amide hydrogen exchange rates in rabbit muscle aldolase, a homotetramer with M(r) = 157,000. Following a period of deuterium exchange, the partially deuterated protein was proteolytically fragmented into peptides whose deuterium contents were determined by directly coupled HPLC fast atom bombardment mass spectrometry. Hydrogen exchange rates were determined for amide hydrogens located in short segments derived from 85% of the aldolase backbone. Isotopic exchange rate constants spanning the range from 100 to 0.001 h-1 were determined for the exchange-in times used in this study (2.5 min to 44 h). The exchange rates for amide hydrogens located within short segments differed by as much as 10(4), demonstrating that local structural features dramatically affect the isotopic exchange rates in large proteins. A high level of correlation between the slowing of hydrogen exchange and intramolecular hydrogen bonding in aldolase was found. An exception to this correlation occurs at the subunit interface, where the amide hydrogens in one peptide segment with few amide hydrogen bonds have slower exchange rates than expected, suggesting that the amide hydrogens in this region are effectively shielded from the deuterated solvent. Isotope patterns observed for most peptides were binomial, indicating that hydrogen exchange proceeds through the EX2 mechanism (uncorrelated exchange). However, bimodal isotope patterns were found for peptides derived from three short segments of aldolase (including residues 58-64, 279-283, and 326-337), suggesting structural differences in these regions. A high level of correlation was found between crystallographic B-factors and amide hydrogen exchange rates, suggesting an isotopic exchange mechanism involving localized low-amplitude, high-frequency motions that do not require collective motion of many residues. From a methodology viewpoint, these results demonstrate that the combination of protein fragmentation with mass spectrometry is a useful method for determining the rates at which amide hydrogens located over major portions of large proteins undergo isotopic exchange.