AMPK Methods and Protocols

1. Prepare fresh H 2 O and D 2 O HDX buffers to match the AMPK
   protein storage buffer (seeNotes 15and 16 ).


2. 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.


3. 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.

4. Perform the MS/MS experiments on a high-resolution-capa-
   ble LTQ XL™linear ion trap mass spectrometer or a similar
   instrument (seeNote 9).


5. 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).


6. 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.


7. Pass the mixture across an in-house packed pepsin column
   (1 mm
   20 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 ).


8. Capture the digested peptides onto a 1 mm
   10 mm C8 trap
   column and desalt them (total time for digestion and desalting
   is 2.5 min).


9. Separate the peptides across a 1 mm
   50 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.