Localized Hydration in
Lyophilized Myoglobin by Hydrogen–Deuterium
Exchange Mass Spectrometry. 1. Exchange Mapping
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Abstract
The local effects of hydration on myoglobin (Mb) in solid
matrices
containing mannitol or sucrose (1:1 w/w, protein:additive) were mapped
using hydrogen–deuterium exchange with mass spectrometric analysis
(HDX–MS) at 5 °C and compared to solution controls. Solid
powders were exposed to D<sub>2</sub>O(g) at controlled activity (<i>a</i><sub>w</sub>) followed by reconstitution and analysis of
the intact protein and peptides produced by pepsin digestion. HDX
varied with matrix type, <i>a</i><sub>w</sub>, and position
along the protein backbone. HDX was less in sucrose matrices than
in mannitol matrices at all <i>a</i><sub>w</sub> while the
difference in solution was negligible. Differences in HDX in the two
matrices were detectable despite similarities in their bulk water
content. The extent of exchange in solids is proposed as a measure
of the hydration of exchangeable amide groups, as well as protein
conformation and dynamics; pepsin digestion allows these effects to
be mapped with peptide-level resolution