- Electrospray ionization parameters are set as follows: sheath
gas 30 au, auxiliary gas 15 au, spray voltage 3.7 V, capillary
temperature 225C, capillary voltage 25 V, tube lens 95 V,
skimmer 16 V.
- Acquire mass spectrometric data with a resolving power of
70,000 at m/z 400.
- Acquire product ion spectra in a data-dependent mode and
then select the five most abundant ions for the product ion
analysis.
- Convert the MS/MS .raw data files to .mgf files. For peptide
identification, submit the MS/MS data to Mascot (seeNote
21 ).
- We usually consider only those peptides that had a Mascot
score of 20 or greater for the HDX analysis. We recommend
running an MS/MS Mascot search against a decoy (reverse)
sequence to rule out ambiguous identifications (seeNote 22).
- Inspect the MS/MS spectra of all the peptide ions from the
Mascot hits for verifiable fragmentation pattern and pepsin
cleavage preferences [28]. Use only those verifiable peptides
for estimating the sequence coverage.
- Map the unambiguously identified peptides onto the AMPK
heterotrimer sequence to estimate the protein sequence cover-
age across all three subunits.
- Repeat MS/MS experiments with varying AMPK protein con-
centration and quench conditions to optimize sequence cover-
age if necessary (seeNote 23).
For hydrogen/deuterium exchange experiments, follow steps
18 – 29 below.
- Prepare a 125μlof10μM solution of Apo AMPK in H 2 O
HDX buffers and dispense in position A1 of 96-well sample
plate. For each AMPK-ligand complex pairwise comparison,
prepare 125μlof10μM AMPK solution containing each
individual ligand at 10final concentration and dispense it in
position A2. Aliquot 100μlofH 2 O HDX buffer in A3 for
blank measurements (seeNotes 24and 25 ).
- Place an empty 96-well tray inside the robotic station assigned
for sample mixing.
- Aliquot 30μl of ice-cold quench solution in wells for rows A–C
and E of the 96-well quench tray.
- Allow the samples to equilibrate inside the HDX system for
1 h. This incubation time can be adjusted for ligands that bind
with weak affinity to AMPK based on SPR data or reduced for
protein systems that are less stable over long experiment times
(12–14 h) needed for the automated HDX-MS analysis.
Biophysical Studies to Evaluate Protein-Ligand Interactions 39
