Digestion Buffer is run through the sample loop and over the
pepsin columns at an increased flow rate of 400 μL/min. The
washing method should be optimized to ensure that there is
less than 5% carryover of peptides from the previous sample. To
monitor the carryover, one should routinely check the Total
Ion Current chromatogram (TIC; All MS) of the short ~8-min
blank (see Note 7).
7. When a sample is inputted into Compass MS Data Analysis
Software, the data will be presented as a Base Peak Chromatogram (BPC) which normalizes all ions detected to the most
intense peak in the mass spectrum and plots them as a function
of time. This tends to clean up the spectrum; however, when
analyzing the short blank run for peptide carryover, the TIC
trace, which displays all the ions detected and plots them as a
function of time, is useful to determine if carryover is likely to
be a problem (see Fig. 2b for an example).
8. Different MS systems will likely have slightly different optimal
MS parameters to maximize sensitivity of detection of both
precursors and fragments. For our Bruker Imapct HD system,
we have found the following ESI and MS parameters to be
optimal for sensitivity. Capillary voltage (4500 V), plate offset
(500 V), drying N 2 gas (6.5 L/min), Nebulizer gas (0.8 Bar),
Dry temp (200
C). For both MS and MS/MS analysis, data
was collected from an m/z range from 150 to 2000. For
MS/MS analysis, data-dependent analysis was carried out
using a 0.5 s precursor scan, followed by twelve 0.25 s fragment
scans.
9. Once exported as a comma separated values (CSV) file, the
peptide list must be modified to make sure only peptides from
the protein of interest are analyzed. The list is filtered using the
ppm error, with any peptide that is >5 ppm from the average
standard error being filtered out of the data set. The file is now
ready for use in HDExaminer; save the file as a modified
peptide list with the .csv extension.
10. It is critical to appropriately determine the threshold for defining a significant change in deuterium incorporation. Using a
difference in the # of deuterium incorporated will bias towards
long peptides, with using a difference in the % of deuterium
incorporated will bias towards short peptides. For this reason,
we use a combined threshold of differences in both the number
of deuterons incorporated and the percentage of deuterium
incorporated. Critically the difference in incorporation must
also cross a significance threshold of an unpaired student t-test.
Probing Protein-Membrane Interactions Using HDX-MS
483
pepsin columns at an increased flow rate of 400 μL/min. The
washing method should be optimized to ensure that there is
less than 5% carryover of peptides from the previous sample. To
monitor the carryover, one should routinely check the Total
Ion Current chromatogram (TIC; All MS) of the short ~8-min
blank (see Note 7).
7. When a sample is inputted into Compass MS Data Analysis
Software, the data will be presented as a Base Peak Chromatogram (BPC) which normalizes all ions detected to the most
intense peak in the mass spectrum and plots them as a function
of time. This tends to clean up the spectrum; however, when
analyzing the short blank run for peptide carryover, the TIC
trace, which displays all the ions detected and plots them as a
function of time, is useful to determine if carryover is likely to
be a problem (see Fig. 2b for an example).
8. Different MS systems will likely have slightly different optimal
MS parameters to maximize sensitivity of detection of both
precursors and fragments. For our Bruker Imapct HD system,
we have found the following ESI and MS parameters to be
optimal for sensitivity. Capillary voltage (4500 V), plate offset
(500 V), drying N 2 gas (6.5 L/min), Nebulizer gas (0.8 Bar),
Dry temp (200
C). For both MS and MS/MS analysis, data
was collected from an m/z range from 150 to 2000. For
MS/MS analysis, data-dependent analysis was carried out
using a 0.5 s precursor scan, followed by twelve 0.25 s fragment
scans.
9. Once exported as a comma separated values (CSV) file, the
peptide list must be modified to make sure only peptides from
the protein of interest are analyzed. The list is filtered using the
ppm error, with any peptide that is >5 ppm from the average
standard error being filtered out of the data set. The file is now
ready for use in HDExaminer; save the file as a modified
peptide list with the .csv extension.
10. It is critical to appropriately determine the threshold for defining a significant change in deuterium incorporation. Using a
difference in the # of deuterium incorporated will bias towards
long peptides, with using a difference in the % of deuterium
incorporated will bias towards short peptides. For this reason,
we use a combined threshold of differences in both the number
of deuterons incorporated and the percentage of deuterium
incorporated. Critically the difference in incorporation must
also cross a significance threshold of an unpaired student t-test.
Probing Protein-Membrane Interactions Using HDX-MS
483