Extended Data Fig. 3: GAP can be measured as its aniline adduct as it has a unique mass.
From: Dihydroxyacetone phosphate signals glucose availability to mTORC1
a, In cells with normal TPI activity, the DHAP and GAP metabolites elute at similar retention times (11 mins vs 11.5 mins, respectively) but the peaks are separate enough to integrate independently. However, when cells lose TPI expression, the DHAP levels increase by an order of magnitude, and the peak normally integrated to measure GAP is obscured by the broader DHAP peak, which has a shoulder that extends into the GAP peak at 11.5 mins. Because the mass of DHAP and GAP are the same, quantifying them accurately in the context of TPI loss is an analytical challenge. HEK-293T AMPKα DKO TPI dox-off cells were treated with (+Dox) or without dox (-Dox) for 10 days. The media was then replaced with fresh RPMI media containing 5 mM glucose for thirty minutes. Metabolites were extracted and analyzed by HILIC LC/MS. Peak traces are shown for the +TPI (-Dox) condition in black and -TPI (+Dox) in blue, both separately with different y-axis scales, or overlaid to highlight the observed changes in DHAP. b, DHAP and GAP can form aniline adducts that are singly labeled with an m/z value of 244.03803 but only GAP can form an aniline adduct that is doubly labeled with an m/z value of 319.08532. c, As expected, only GAP forms a product with m/z value of 319.08532 when reacted with aniline. 10 μM GAP or DHAP standards were reacted with aniline in the presence of EDC in 80% methanol to yield their respective aniline adducts. The reaction was analyzed by reverse phase LC/MS. d, Metabolites upstream of TPI increase and metabolites downstream of TPI decrease in the absence of TPI expression. HEK-293T AMPKα DKO TPI dox-off cells were incubated in full media (5mM glucose) following 10 days of doxycycline. Half of the extract was used in an aniline labeling reaction and analyzed by reverse phase LC/MS and the other half was analyzed by HILIC LC/MS. These data are an extension of the data shown in Fig. 4b, showing only the unstarved samples. Data are shown as mean ± s.e.m. for n = 3 biologically independent replicates.
