Global metabolite profiling

PDAC cells (5 × 10⁶) were collected in 80% HPLC-grade methanol (4 methanol/1 water, v/v) and centrifuged. The supernatant was transferred to a tube and dried under vacuum. The precipitate was dissolved in 50 μl of 80% HPLC-grade methanol and protein level was determined. We applied ultrahigh-performance liquid chromatography coupled with a TSQ Quantiva mass spectrometer (HPLC-MS, Thermo Scientific) to globally profile the metabolites. Metabolite identification and data processing were accomplished by TraceFinder 3.2 (Thermo).

For acylcarnitine analysis, PDAC cells (5 × 10⁶) were collected in 80% HPLC-grade methanol, sonicated, and centrifuged. The supernatant (3 μl) was mixed with 100 μl of ²H-labeled carnitine standard working solution dissolve in 2 ml HPLC-grade methanol (Cambridge Isotope Laboratories, Inc.). The mixture was centrifuged and the supernatant was collected and dried with N₂ at 50 °C. The sample was resuspended in 60 μl of acetyl chloride/1-butanol mixture (1:9, v/v) and incubated at 65 °C for 20 min. The derived sample was dried with N₂ and dissolved in 100 μl of 80% acetonitrile before being subject to an AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA). Data acquisition was conducted with Analyst 1.6 (AB Sciex) and metabolite identification and data processing were accomplished using ChemoView 2.0.2 (AB Sciex).

For ¹³C-U₆-glucose metabolism analysis, PDAC cells seed for 24 h were cultured in medium with ¹³C-U₆-glucose (25 mM) for another 48 h. Then the medium was removed and cells were exposed to ¹³C-U₆-glucose again for 2 h. The sample prepared as mentioned above was subject to HPLC-MS analysis. ¹³C-U₆-glucose tracing was also accomplished using gas chromatography-mass spectrometry (GC-MS, Thermo Fisher Scientific Trace 1300). Cells exposed to ¹³C-U₆-glucose were collected in extractant consisting of HPLC-grade methanol, tert-Butyl methyl ether (Sigma-Aldrich), and H₂O (1/2/1, v/v/v) and extracted by vortex for 15 min. After centrifugation, both ether and methanol layers were, respectively, collected for analysis of fatty acid flux and TCA intermediate metabolite flux. The ether extract was dried by N₂ and derived in 2% H₂SO₄/methanol solution (1/49, v/v) for 1 h at 50 °C. The derivatives extracted by saturated NaCl solution and hexane were dissolved in hexane and subject to GC-MS (Thermo Fisher). The methanol extract was dried under vacuum and derived in 2% (w/v) methoxyamine hydrochloride in pyridine for 1 h at 37 °C, followed by silylation in 30 μl of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide plus 1% of tert-butyldimethylchlorosilane (Regis Technologies) for 30 min at 45 °C. The resultant derivatives were subject to GC-MS.