- Open the abdomen to expose the organs in the peritoneal
cavity and tightly clamp the edge of liver with the prefreezed
clamp for 20 s. - Homogenate some 50 mg of frozen liver using an
electrical disperser in 1 mL of methanol containing IS1
(1:200;seeNote 41). - Extract ATP, ADP, and AMP as described insteps 4– 11 of
Subheading3.4.
Measure the Levels of AMP, ADP, and ATP Using CE-MS- Maintain the fused-silica capillary at 20C prior to use (see
Note 42). - Couple CE unit with TOF-MS unit by passing the sheath
liquid through the coaxial sheath at 10μL/min for 30 min. - Run CE-TOF-MS at anion mode and analyze the levels of
adenylates. In detail, the fused-silica capillary is first filled
with ammonium acetate solution and then preconditioned
with the electrolyte containing phosphate buffer. At the begin-
ning of each run, the capillary is washed by electrolyte contain-
ing phosphate buffer. Sample is injected by Agilent 1100 series
pump at a pressure of 50 mbar for 25 s and is driven by a
constant voltage at 30 kV (for 40 min). The fragmentor volt-
age of MS unit at three directions is set at 125, 50, and 650 V,
respectively. The voltage for ionization is set at 3500 V, with a
drying gas flow at 7 L/min at 300C. The separation is assisted
by a nebulizer at 15 mbar pressure. The isotope of the depro-
tonated acetic acid (m/z60.0172) and 2,2-difluoromethoxy
phosphazene plus deprotonated acetic acid (m/z680.03554)
are used as the reference masses. The scanning range is
50–1000m/z, and the scan rate is 1.5 spectra/s.
4 Notes
- Dialysis of FBS is not necessary, since the glucose brought by
10% (v/v) FBS is not sufficient to affect the activation of AMPK
under glucose starvation.
2.LAMTOR1/orAXIN/MEFs are generated by infecting
SV40 T-immortalizedLAMTOR1F/ForAXINF/FMEFs with
adenovirus expressing Cre recombinase for 12 h. To avoid the
elevated basal levels of p-Thr-172-AMPKαin the infected cells,
we strongly recommend that cells be incubated in fresh
DMEM for another 8–10 h before further treatments. - According to our experience, the genomes ofLAMTOR1/
MEFs are unstable. We therefore recommend generating
knockout MEFs fresh for each experiment.
406 Chen-Song Zhang et al.