3. Run a standard protein (e.g., horse heart myoglobin or rabbit
muscle phosphorylase B) to verify the LC separation and protease column activity. This standard protein used for system
quality control must be different from the protein of interest.
Always keep in mind that the protein must be prepared in an
acidic buffer as the digestion is done using aspartic proteases.
Accidental injection of neutral pH buffer will quickly and
irreversibly inhibit pepsin. Other proteases mentioned here
are more robust in this regard and can tolerate elevated pH
temporarily. Typically prepare 30 pmol of myoglobin in 100 μl
of 250 mM glycine-Cl buffer pH 2.3.
4. Run two blank injections (100 μl of pure quench buffer) to
clean the system and to ensure proper and stable LC conditions
(see Note 14).
5. Mix the sample with the quench buffer to get the sample
volume and concentration as planned for subsequent
HDX-MS experiment (see Note 15). Hence, 50 μl of
500 mM glycine-Cl buffer pH 2.3 with 50 μl of 2 μM protein
solution in 20 mM HEPES, pH 7.4, 150 mM NaCl.
6. Inject the sample directly or subject it to a freeze–thaw cycle if
you plan to collect aliquots by freezing in liquid nitrogen.
7. Wait for desired time (3 min) until digestion and desalting are
finished (see Note 16) and switch the second LC valve so that
the trap column is now in the path of gradient elution and the
peptides will continuously elute on the analytical column.
Together with the switch, also start the gradient (see Note
17) and collect the MS data.
8. Process the LC-MS/MS file using the instrument vendorspecific software and generate input for the search engine (see
Note 18).
9. Run a database search using the input file from the previous
step. Use a custom-made database containing the sequence of
the protein of interest and the protease(s) used and eventually
other proteins present in the sample. Do not set any digestion
preferences, nor taxonomy filtering. Include possible relevant
modifications (fixed or variable) that may occur in the protein.
Set the mass tolerance on precursor and fragments according to
your instrument performance. Use scoring routines to discard
improbable matches. It is also advisable to run the LC-MS/MS
analysis 3–4 times and only use peptide identifications that
occur in the majority of the analyses.
10. Copy or export the search result (list of identified peptides) to a
spreadsheet editor (e.g., Microsoft Excel) and extract the Peptide Start and End columns (peptide limits). Use this in a
simple text file to visualize the coverage map using DrawMap
HDX-MS of Protein-DNA Complexes
201
muscle phosphorylase B) to verify the LC separation and protease column activity. This standard protein used for system
quality control must be different from the protein of interest.
Always keep in mind that the protein must be prepared in an
acidic buffer as the digestion is done using aspartic proteases.
Accidental injection of neutral pH buffer will quickly and
irreversibly inhibit pepsin. Other proteases mentioned here
are more robust in this regard and can tolerate elevated pH
temporarily. Typically prepare 30 pmol of myoglobin in 100 μl
of 250 mM glycine-Cl buffer pH 2.3.
4. Run two blank injections (100 μl of pure quench buffer) to
clean the system and to ensure proper and stable LC conditions
(see Note 14).
5. Mix the sample with the quench buffer to get the sample
volume and concentration as planned for subsequent
HDX-MS experiment (see Note 15). Hence, 50 μl of
500 mM glycine-Cl buffer pH 2.3 with 50 μl of 2 μM protein
solution in 20 mM HEPES, pH 7.4, 150 mM NaCl.
6. Inject the sample directly or subject it to a freeze–thaw cycle if
you plan to collect aliquots by freezing in liquid nitrogen.
7. Wait for desired time (3 min) until digestion and desalting are
finished (see Note 16) and switch the second LC valve so that
the trap column is now in the path of gradient elution and the
peptides will continuously elute on the analytical column.
Together with the switch, also start the gradient (see Note
17) and collect the MS data.
8. Process the LC-MS/MS file using the instrument vendorspecific software and generate input for the search engine (see
Note 18).
9. Run a database search using the input file from the previous
step. Use a custom-made database containing the sequence of
the protein of interest and the protease(s) used and eventually
other proteins present in the sample. Do not set any digestion
preferences, nor taxonomy filtering. Include possible relevant
modifications (fixed or variable) that may occur in the protein.
Set the mass tolerance on precursor and fragments according to
your instrument performance. Use scoring routines to discard
improbable matches. It is also advisable to run the LC-MS/MS
analysis 3–4 times and only use peptide identifications that
occur in the majority of the analyses.
10. Copy or export the search result (list of identified peptides) to a
spreadsheet editor (e.g., Microsoft Excel) and extract the Peptide Start and End columns (peptide limits). Use this in a
simple text file to visualize the coverage map using DrawMap
HDX-MS of Protein-DNA Complexes
201