Liquid chromatography-mass spectrometry (LC-MS)
Mice brain tissue samples were isolated and immediately snap-frozen in
liquid nitrogen. 50-100 mg of frozen tissue samples were taken for
further analysis. The samples were lysed in water: methanol (60: 40)
solvent system, 0.5 ml solvent for 100 mg tissue using an ultrasonic
homogenizer (sonics VCX750, 40% amplitude, 3 pulses of 7 sec each at
4°C). The samples were incubated at 4°C for 30 minutes with vertexing
vigorously after every 10 minutes interval, followed by centrifugation
at 14000g for 20 minutes at 4°C. The supernatant was collected, and a
syringe filter was used to filter out any remaining debris or
impurities. Samples were taken for further LCMS quantification of ATP,
ADP, AMP, NAD+, and NADH. The remaining pellet was used to quantify the
protein using the Bradford method, and the quantified metabolites were
further normalized with total protein. The LCMS analysis conditions are
listed in Table 1.