Localized Hydration in
Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry.
2. Exchange Kinetics
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Abstract
Solid-state hydrogen–deuterium exchange with mass
spectrometric analysis (ssHDX) is a promising method for characterizing
proteins in amorphous solids. Though analysis of HDX kinetics is informative
and well-established in solution, application of these methods to
solid samples is complicated by possible heterogeneities in the solid.
The studies reported here provide a detailed analysis of the kinetics
of hydration and ssHDX for equine myoglobin (Mb) in solid matrices
containing sucrose or mannitol. Water sorption was rapid relative
to ssHDX, indicating that ssHDX kinetics was not limited by bulk water
transport. Deuterium uptake in solids was well-characterized by a
biexponential model; values for regression parameters provided insight
into differences between the two solid matrices. Analysis of the widths
of peptide mass envelopes revealed that, in solution, an apparent
EX2 mechanism prevails, consistent with native conformation of the
protein. In contrast, in mannitol-containing samples, a smaller non-native
subpopulation exchanges by an EX1-like mechanism. Together, the results
indicate that the analysis of ssHDX kinetic data and of the widths
of peptide mass envelopes is useful in screening solid formulations
of protein drugs for the presence of non-native species that cannot
be detected by amide I FTIR