Hydrogen Deuterium Exchange in the Frozen State

evidence of H/D exchange between LDH and
deuterium solvent in the frozen state is shown in Fig. 2. In
Fig. 1 Two protocols used for
HX labeling (Protocol A) in the
frozen state, and (Protocol B) after
subjection to F/T cycles. The time
for each step is designated
underneath.
1182 Zhang, Qi, Singh and Fernandez
this experiment, 0.1 mg/ml LDH was frozen in 90%
deuterium buffer using liquid N2, and then stored in a
−10°C freezer for a period of time ranging from 10 min up
to 4 days. The degree of deuterium labeling was measured
by the mass increase relative to unlabeled LDH by mass
spectrometry. As shown in Fig. 2, the molecular mass of
LDH increased with incubation time in the −10°C freezer,
demonstrating there was continuous incorporation of
deuterium into LDH within the experimental time range
in the frozen state. The deuteration level calculated by
equation (1) increased from 21.8% to 68.3% with the
labeling time increasing from 10 min to 4 day. For studies
under variable protein and solution conditions, an intermediate
incubation time of 12 h at −10°C was selected for
LDH conformation analysis in the frozen state for two
reasons. First, the observed LDH mass increase from
10 min to 12 h in Fig. 2 suggested that a significant
fraction of deuterium labeling was achieved in the frozen
sample state during the 12 h incubation, which reduced
the effect of H/D exchange in the solution state following
LDH sample and D2O labeling buffer mixing. Second, the
deuteration level of LDH after 12 h incubation was not
particularly high (38.6%) relative to the fully unfolded
control (100%), and it favored the resolution of labeling
peaks for native and partially unfolded LDH in the frozen
state shown below.