run. Using the set of coordinates consisting of peptide sequence, its
mass-over-charge (m/z) value(s) (see Note 31), and LC retention
time, individual features can be located in the partially deuterated
samples. In a subsequent step, information about the number of
deuterons carried by individual peptides is extracted. Two major
approaches to deuteration readout rely either on centroid readout
or on deconvolution and theoretical fitting [36]. Finally, this information is presented in several graphical ways to visualize the effects
monitored by the HDX-MS experiment.
3.5.1 Workflow A:
(Largely Automated) Data
Interpretation Using DeutEx
1. From an MS/MS search engine, e.g., MASCOT (here utilized
by Bruker’s ProteinScape), export the search result in the form
of a csv file (see Note 32).
2. Prepare a separate simple text file containing the sequence of
the studied protein in FASTA format.
3. Open DeutEx and go to Analysis>Compose digest file from MS.
New window will appear. In this window, upload your protein
sequence as a FASTA file, then csv file containing the list of
identified peptides from MASCOT and an LC-MS run of a
non-deuterated sample exported into simple text files (each MS
scan corresponding to one file) containing a list of m/z values
and their intensities (see Note 33).
4. Go to Digest, click Create from report and then Extract scan/
charge limits. Each peptide will have its retention limits automatically identified, and extracted ion chromatograms will be
drawn for individual charge states (m/z values). User can manually inspect the assignments and either manually correct or
completely reject them. Once finished, the result should be
exported into a text file that serves as a lookup file for an
automated assignment of HDX data for the deuterated samples. Close the “Compose digest from MS window.”
5. Go to Analysis>Open Analysis directory. It is advisable to
pre-compile data for each state into a separate folder (which
contains exported LC-MS data in a txt format, with appropriate
fasta sequence and digest file—the list of peptides created in
step 4). However, the data can also be compiled in DeutEx
using the “Compose analysis” function.
6. Once all data are opened, check the settings for mass accuracies
and filtering under the options menu and run the calculation.
Typically, it lasts from seconds to a few minutes, depending on
the number of conditions, number of peptides, and size of the
exported data on a common laptop/office computer. When
calculation is finished, run filtering, which tries to automatically
remove incorrect assignments. Then look through the assignments manually, check the data, and correct any misassignments, if needed.
HDX-MS of Protein-DNA Complexes
207
mass-over-charge (m/z) value(s) (see Note 31), and LC retention
time, individual features can be located in the partially deuterated
samples. In a subsequent step, information about the number of
deuterons carried by individual peptides is extracted. Two major
approaches to deuteration readout rely either on centroid readout
or on deconvolution and theoretical fitting [36]. Finally, this information is presented in several graphical ways to visualize the effects
monitored by the HDX-MS experiment.
3.5.1 Workflow A:
(Largely Automated) Data
Interpretation Using DeutEx
1. From an MS/MS search engine, e.g., MASCOT (here utilized
by Bruker’s ProteinScape), export the search result in the form
of a csv file (see Note 32).
2. Prepare a separate simple text file containing the sequence of
the studied protein in FASTA format.
3. Open DeutEx and go to Analysis>Compose digest file from MS.
New window will appear. In this window, upload your protein
sequence as a FASTA file, then csv file containing the list of
identified peptides from MASCOT and an LC-MS run of a
non-deuterated sample exported into simple text files (each MS
scan corresponding to one file) containing a list of m/z values
and their intensities (see Note 33).
4. Go to Digest, click Create from report and then Extract scan/
charge limits. Each peptide will have its retention limits automatically identified, and extracted ion chromatograms will be
drawn for individual charge states (m/z values). User can manually inspect the assignments and either manually correct or
completely reject them. Once finished, the result should be
exported into a text file that serves as a lookup file for an
automated assignment of HDX data for the deuterated samples. Close the “Compose digest from MS window.”
5. Go to Analysis>Open Analysis directory. It is advisable to
pre-compile data for each state into a separate folder (which
contains exported LC-MS data in a txt format, with appropriate
fasta sequence and digest file—the list of peptides created in
step 4). However, the data can also be compiled in DeutEx
using the “Compose analysis” function.
6. Once all data are opened, check the settings for mass accuracies
and filtering under the options menu and run the calculation.
Typically, it lasts from seconds to a few minutes, depending on
the number of conditions, number of peptides, and size of the
exported data on a common laptop/office computer. When
calculation is finished, run filtering, which tries to automatically
remove incorrect assignments. Then look through the assignments manually, check the data, and correct any misassignments, if needed.
HDX-MS of Protein-DNA Complexes
207