- Prepare fresh H 2 O and D 2 O HDX buffers to match the AMPK
protein storage buffer (seeNotes 15and 16 ). - Prepare 125μl of each sample (Apo protein or ligand complex)
at a final working concentration of 10μMinH 2 O HDX buffer.
Initiate complex formation by incubating the AMPK protein
sample with tenfold molar excess (100μM final concentration)
of ligand(s) from DMSO stock solutions or equal volume of
100% DMSO (Apo) and incubate for 1 h on ice (or desired
temperature) before subjecting to HDX analysis. - Before running a HDX experiment (MS^1 data) to measure
deuterium uptake kinetics, it is a prerequisite to generate a list
of peptic peptides (generated by treatment with pepsin) cover-
ing all three AMPK subunits using tandem MS (MS/MS or
MS^2 ) under identical sample, chromatographic, and MS con-
ditions needed for HDX. Scan this peptide set against HDX
datasets (see below) to estimate the deuteration profiles for
each peptide based on its monoisotopic mass and deuterium
uptake over time. For AMPK heterotrimers (134 kDa), we
followed over 500 peptides.
For peptide identification and tandem MS, followsteps 4– 17 below.
- Perform the MS/MS experiments on a high-resolution-capa-
ble LTQ XL™linear ion trap mass spectrometer or a similar
instrument (seeNote 9). - Prepare a 10μM solution of AMPK in H 2 O HDX buffers. For
protein-protein complex studies, it is advisable to prepare the
AMPK sample in the presence of other interacting partners to
optimize pepsin digestion efficiency and sequence coverage in
the context of complex (seeNote 17). - Dilute 4μlof10μM AMPK solution with 16μl of AMPK
HDX H 2 O buffer and then mix with 30μl of quench solution
(seeNote 18). Inject 50 μl into the LC/MS for protease
digestion, peptide trapping, and gradient elution. - Pass the mixture across an in-house packed pepsin column
(1 mm20 mm) at 50μl/min. We usually place the pepsin
column inside a temperature control chamber held at 15Cto
allow for optimal pepsin digestion activity (seeNotes 19and
20 ). - Capture the digested peptides onto a 1 mm10 mm C8 trap
column and desalt them (total time for digestion and desalting
is 2.5 min). - Separate the peptides across a 1 mm50 mm C18 analytical
column with a 75- min linear gradient (5–50% ACN and 0.3%
FA over 60 min initially; and then increased to 100% ACN,
0.3% FA for 15 min, and finally reduced to 5% ACN, 0.3% FA
within 1 min).
38 Ravi G. Kurumbail et al.