4. Establishing the appropriate comparisons between conditions,
as needed. This is done by computing the difference between
both the % deuteration and # of deuterons for the conditions
that are to be compared. In the Akt study, comparisons were
made between the apo and membrane conditions.
5. Determining the thresholds for significance for differences in
deuterium incorporation. Our standard threshold requires
three specific criteria be met: >7% change in deuterium incorporation at any time point, >0.4 Da difference in deuterium
incorporation, and an unpaired student t-test value of
P < 0.01% (see Note 10 for more details).
3.3.5 Data Analysis
and Presentation
of Deuterium Incorporation
and Differences Between
Conditions
Critical to the proper analysis of the deuterium exchange information is the generation of the appropriate visualization of the data.
This processing includes:
1. Analysis of the H/D incorporation heatmap. Deuterium incorporation over the entire protein can be viewed as a heatmap,
where the level of deuterium incorporation, as indicated by
coloring according to the percentage of deuterium exchange,
can be viewed at every time point for all conditions tested
(Fig. 3b). This data can also be presented in what is termed as
a butterfly plot (Fig. 3c), where the %D is graphed according to
the sequence, with SD information included. Two conditions
can be viewed on a butterfly plot at the same time, which may
reveal differences in exchange. Very localized regions of
exchange can be determined using overlapping fragments;
however, careful interpretation of overlapping fragments is
critical. Any differences observed in this analysis must be verified at the single peptide level (see below).
2. Generation of deuterium incorporation timecourses. The deuterium incorporation of every peptide analyzed should be
graphed for all conditions measured, with error bars describing
standard deviation included (Figs. 2d and 3f). These graphs are
directly generated in HDExaminer and can also be generated in
GraphPad Prism Software downstream of the excel analysis.
3. Analysis of differences in deuterium incorporation. The differences in deuterium incorporation (either %D or #D) summed
across all time points can be visualized over peptide sequence
(Fig. 3d). One of the easiest ways to interpret the molecular
consequences of the differences in exchange is mapping
changes in deuterium incorporation on a structural model of
the protein of interest if it is available (Fig. 3e), with the
magnitude of the difference in deuterium incorporation indicated by different colors. This can be done by mapping the
differences at any time point on a single model, or mapping the
difference at each time point on the model (i.e., four different
478
Jordan T. B. Stariha et al.
as needed. This is done by computing the difference between
both the % deuteration and # of deuterons for the conditions
that are to be compared. In the Akt study, comparisons were
made between the apo and membrane conditions.
5. Determining the thresholds for significance for differences in
deuterium incorporation. Our standard threshold requires
three specific criteria be met: >7% change in deuterium incorporation at any time point, >0.4 Da difference in deuterium
incorporation, and an unpaired student t-test value of
P < 0.01% (see Note 10 for more details).
3.3.5 Data Analysis
and Presentation
of Deuterium Incorporation
and Differences Between
Conditions
Critical to the proper analysis of the deuterium exchange information is the generation of the appropriate visualization of the data.
This processing includes:
1. Analysis of the H/D incorporation heatmap. Deuterium incorporation over the entire protein can be viewed as a heatmap,
where the level of deuterium incorporation, as indicated by
coloring according to the percentage of deuterium exchange,
can be viewed at every time point for all conditions tested
(Fig. 3b). This data can also be presented in what is termed as
a butterfly plot (Fig. 3c), where the %D is graphed according to
the sequence, with SD information included. Two conditions
can be viewed on a butterfly plot at the same time, which may
reveal differences in exchange. Very localized regions of
exchange can be determined using overlapping fragments;
however, careful interpretation of overlapping fragments is
critical. Any differences observed in this analysis must be verified at the single peptide level (see below).
2. Generation of deuterium incorporation timecourses. The deuterium incorporation of every peptide analyzed should be
graphed for all conditions measured, with error bars describing
standard deviation included (Figs. 2d and 3f). These graphs are
directly generated in HDExaminer and can also be generated in
GraphPad Prism Software downstream of the excel analysis.
3. Analysis of differences in deuterium incorporation. The differences in deuterium incorporation (either %D or #D) summed
across all time points can be visualized over peptide sequence
(Fig. 3d). One of the easiest ways to interpret the molecular
consequences of the differences in exchange is mapping
changes in deuterium incorporation on a structural model of
the protein of interest if it is available (Fig. 3e), with the
magnitude of the difference in deuterium incorporation indicated by different colors. This can be done by mapping the
differences at any time point on a single model, or mapping the
difference at each time point on the model (i.e., four different
478
Jordan T. B. Stariha et al.