sequence coverage. For Akt, the final peptide list covered ~95% of
the protein (Fig. 3a). The final product of this analysis is a comma
separated values (CSV) file, containing the sequence, charge state,
ppm mass error, and retention time. The file is modified prior to
uploading to HDExaminer to remove peptide hits from other
proteins as well as peptides with large ppm errors (see Note 9).
3.3.2 Measuring
Deuterium Incorporation
with HDExaminer
HDExaminer (Sierra Analytics) is a commercial software package
used for calculating deuterium incorporation that is compatible
with multiple MS vendor formats. This software uses the mass
centroid as well as envelope-shape information of the isotopic distributions corresponding to each peptide and displays deuterium
incorporation values in terms of both % deuteration and number of
deuterons (Da). It is essential to manually verify the deuterium
incorporation of every peptide at every time point and replicate,
to make sure the peak is accurately fit. It is very important to
examine the shape of the deuterium incorporation distribution, as
it provides deuterium occupancy information corresponding to
specific residues within peptides, as well as details on the mechanism
of deuterium incorporation (EX2 and EX1 kinetics; [36, 37]). This
data is generally lost when only looking at the mass centroid. Readers are advised to consult the following reviews for more details on
data analysis [38, 39].
3.3.3 Starting a Project
on HDExaminer
Starting a new HDExaminer project requires three key components
which are added to the software by dragging the files into the
software in the following order:
1. A text file (.txt extension) containing the FASTA sequence for
the protein of interest. When performing mutational analysis,
the appropriate mutations can be applied to the sequence.
2. The modified peptide list of the protein of interest, generated
by PEAKS7. This list is exported from PEAKS7 and modified
to include only the peptides from the protein of interest that are
within a certain ppm error (average ppm Æ 5; see Note 9).
3. A non-deuterated MS data file for the protein of interest. This
software searches the datafile for the presence of peptides identified in PEAKS7. At this step, the initial peptide list is curated
to only include peptides that will be trackable throughout the
dataset, with this decision based on peptide’s signal/noise and
level of interference from overlapping peptides (further details
described below). The peptides remaining are used as an
undeuterated reference point for the calculation of deuterium
incorporation for all other samples.
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the protein (Fig. 3a). The final product of this analysis is a comma
separated values (CSV) file, containing the sequence, charge state,
ppm mass error, and retention time. The file is modified prior to
uploading to HDExaminer to remove peptide hits from other
proteins as well as peptides with large ppm errors (see Note 9).
3.3.2 Measuring
Deuterium Incorporation
with HDExaminer
HDExaminer (Sierra Analytics) is a commercial software package
used for calculating deuterium incorporation that is compatible
with multiple MS vendor formats. This software uses the mass
centroid as well as envelope-shape information of the isotopic distributions corresponding to each peptide and displays deuterium
incorporation values in terms of both % deuteration and number of
deuterons (Da). It is essential to manually verify the deuterium
incorporation of every peptide at every time point and replicate,
to make sure the peak is accurately fit. It is very important to
examine the shape of the deuterium incorporation distribution, as
it provides deuterium occupancy information corresponding to
specific residues within peptides, as well as details on the mechanism
of deuterium incorporation (EX2 and EX1 kinetics; [36, 37]). This
data is generally lost when only looking at the mass centroid. Readers are advised to consult the following reviews for more details on
data analysis [38, 39].
3.3.3 Starting a Project
on HDExaminer
Starting a new HDExaminer project requires three key components
which are added to the software by dragging the files into the
software in the following order:
1. A text file (.txt extension) containing the FASTA sequence for
the protein of interest. When performing mutational analysis,
the appropriate mutations can be applied to the sequence.
2. The modified peptide list of the protein of interest, generated
by PEAKS7. This list is exported from PEAKS7 and modified
to include only the peptides from the protein of interest that are
within a certain ppm error (average ppm Æ 5; see Note 9).
3. A non-deuterated MS data file for the protein of interest. This
software searches the datafile for the presence of peptides identified in PEAKS7. At this step, the initial peptide list is curated
to only include peptides that will be trackable throughout the
dataset, with this decision based on peptide’s signal/noise and
level of interference from overlapping peptides (further details
described below). The peptides remaining are used as an
undeuterated reference point for the calculation of deuterium
incorporation for all other samples.
476
Jordan T. B. Stariha et al.