3.3.4 Data Processing
of Deuterated Samples
Drawing meaningful and accurate conclusions from HDX-MS
experiments requires that the data be manually processed and
curated. Data processing is performed in two stages:
1. Processing data in HDExaminer. Initial analysis of the
non-deuterated file can speed up analysis during later steps by
reducing the number of overlapping peptides in areas that are
highly concentrated and filling in any essential gaps in the data
by searching multiple charge states. The final peptide list for
Akt is shown in Fig. 3a. Examples of the raw deuterium incorporation data for two selected peptides in Akt in the presence
and absence of membrane are shown in Fig. 2c. Every peptide
needs to be scrutinized on HDExaminer to look for the following:t
(a) Correct charge state;
(b) Correct retention time (provided by PEAKS7). The
selected range can be refined to exclude contaminating
peaks. Tightening the range will help remove coeluting
peptides, but may also reduce the peptide signal.
(c) Appropriate selection of isotopic distribution;
(d) Carryover (see Note 6).
(e) Isotopic distributions from co-eluting peptides of similar
m/z that overlap/mix with the selected distribution, and
may therefore skew the data.
(f) If more than one protein is present in the sample, ensure
that the selected isotopic distribution is from the appropriate protein and not its binding partner. This may occur
if the binding partner is present in a much higher
concentration.
2. Further processing of data in excel. Once the HDExaminer file
has been scrutinized and curated, it can be exported as an excel
document. This document allows for analysis of the raw data
from HDExaminer in order to produce interpretable data. This
stage of processing includes the following steps:
3. Generation of the average and standard deviation of deuterium
incorporation for all peptides analyzed. Any outliers (high SD,
or nonstandard deuterium incorporation curves (e.g., deuterium incorporation decreasing over time)) should be manually
inspected again in HDExaminer. When this is finalized, the
final data should include the sequence of every peptide, the
charge state, the level of deuterium incorporation, and the
standard deviation for the triplicate measurement. This data
should be included as supplemental data for any publication to
allow for detailed review and analysis.
Probing Protein-Membrane Interactions Using HDX-MS
477
of Deuterated Samples
Drawing meaningful and accurate conclusions from HDX-MS
experiments requires that the data be manually processed and
curated. Data processing is performed in two stages:
1. Processing data in HDExaminer. Initial analysis of the
non-deuterated file can speed up analysis during later steps by
reducing the number of overlapping peptides in areas that are
highly concentrated and filling in any essential gaps in the data
by searching multiple charge states. The final peptide list for
Akt is shown in Fig. 3a. Examples of the raw deuterium incorporation data for two selected peptides in Akt in the presence
and absence of membrane are shown in Fig. 2c. Every peptide
needs to be scrutinized on HDExaminer to look for the following:t
(a) Correct charge state;
(b) Correct retention time (provided by PEAKS7). The
selected range can be refined to exclude contaminating
peaks. Tightening the range will help remove coeluting
peptides, but may also reduce the peptide signal.
(c) Appropriate selection of isotopic distribution;
(d) Carryover (see Note 6).
(e) Isotopic distributions from co-eluting peptides of similar
m/z that overlap/mix with the selected distribution, and
may therefore skew the data.
(f) If more than one protein is present in the sample, ensure
that the selected isotopic distribution is from the appropriate protein and not its binding partner. This may occur
if the binding partner is present in a much higher
concentration.
2. Further processing of data in excel. Once the HDExaminer file
has been scrutinized and curated, it can be exported as an excel
document. This document allows for analysis of the raw data
from HDExaminer in order to produce interpretable data. This
stage of processing includes the following steps:
3. Generation of the average and standard deviation of deuterium
incorporation for all peptides analyzed. Any outliers (high SD,
or nonstandard deuterium incorporation curves (e.g., deuterium incorporation decreasing over time)) should be manually
inspected again in HDExaminer. When this is finalized, the
final data should include the sequence of every peptide, the
charge state, the level of deuterium incorporation, and the
standard deviation for the triplicate measurement. This data
should be included as supplemental data for any publication to
allow for detailed review and analysis.
Probing Protein-Membrane Interactions Using HDX-MS
477