Methoxyamine hcl

All three compounds could be separated and detected after derivatization with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) in pyridine (Sigma-Aldrich, USA). Samples were snap frozen in liquid nitrogen and then freeze-dried overnight. The resultant powder was resuspended in 50 μL 240 mM methoxyamine-HCl (Sigma-Aldrich, USA) in pyridine. After incubation at 65°C for 50 minutes, 80 μL of MSTFA was added and the samples were incubated at 65°C for a further 50 minutes. Samples were centrifuged at 10 000 x g for 10 mins, and GC-MS separation was performed with a HP5-MS column (Agilent Technologies, USA) using the following program: Carrier gas helium at 30mL per minute. Oven program: 1 mL/min at 100°C, hold for 0.2 min then ramp at 10°C/min to 250°C and hold for 10 min. Injector temperature: 280°C. Injection volume: 1 μL. Products were detected by selected ion monitoring for DHAP (m/z ions 400, 315, 299, 73), G3P (m/z ions 357, 299, 73) and glycerol (m/z ions 205, 147, 73).

All derivatization steps were carried out in a fume hood. In order to derivatize proteinogenic amino acids, organic acids and glycolytic intermediates for GC-MS analysis, the dried extract was incubated at 95 °C in open tubes in order to remove any residual moisture in the samples. The dried extract was solubilized in 40 μl of 2% methoxyamine HCL in pyridine (Sigma-Aldrich, Dorset,UK) followed by 60 min incubation at 60 °C and subsequently 60 μl N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1% (w/v) tertbutyldimethyl-chlorosilane (TBDMSCI) (Sigma-Aldrich, Dorset, UK) derivatization reagent was added. The suspension was incubated for an hour at 60 °C in a well-sealed tube to prevent evaporation. Finally the samples were centrifuged at 13,000 rpm for 5 min and the clear supernatant was transferred to a chromatography vial with a glass insert (Thermo Fisher, Scientific, Chromacol, Hertfordshire, UK) and proceeded immediately to GC-MS analysis.

Methoxyamine HCl, fatty acid methyl ester (C7–C30, FAMEs) standards, pyridine, and anhydrous sodium sulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA). MSTFA (N-methyl-N(trimethylsilyl)trifluoroacetamide) with 1% (vol/vol) trimethylchlorosilane (MSTFA, with 1% TMCS), methanol (Optima LC-MS), acetonitrile (Optima LC-MS), hexane, dichloromethane, chloroform, and acetone were purchased from Thermo-Fisher Scientific (FairLawn, NJ, USA). Ultrapure water was produced by a Mill-Q Reference system equipped with a LC-MS Pak filter (Millipore, Billerica, MA).

12-[[(cyclohexyl-am[42]ino) carbonyl] amino]-dodecanoic acid (CUDA) at a concentration of 50 ppb was used as an internal standard for the UHPLC-MS analysis. For the GC–MS analysis, the derivatization reagent BSTFA (O-bis (trimethyl-silyl) trifluoroacetamide, Sigma-Aldrich, USA) was used. The methoxy-amine (MOX) reagent was 2% methoxy-amine-HCl in pyridine (Sigma-Aldrich, USA). For the NMR analysis, trimethyl-silyl-propane-sulfonic acid (DSS) was used as reference, and deuterium oxide (D₂O) as a solvent.

Rotenone, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTFA), 3-nitropropionic acid (3-NPA), malonate, 2,6-dichlorophenolindophenol, decylubiquinone, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), phenazine methosulfate (PMS), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP),[¹³C₅,¹⁵N₁]glutamate, methoxyamine HCl, diisopropylethylamine (DIPEA), pentafluorobenzyl bromide, 1,1,1,3,3,3-hexafluoro-2-propanol, dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescin diacetate, N-acetylcysteine, propidium iodide (PI), succinate, [¹³C₄]succinate, [¹³C₃]lactate, [¹³C₆]citrate, and [¹³C₅,¹⁵N₂]glutamine were purchased from Sigma-Aldrich. LND was purchased from Santa Cruz Biotechnology. Optima LC-MS grade water, methanol, acetonitrile, and isopropanol were purchased from Thermo Fisher Scientific (Waltham, MA). [¹³C₄]Fumarate and glutathione ([¹³C₂,¹⁵N₁]glycine) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA).

Cells at ~80% confluency were washed twice in cold saline solution (NaCl, 0.9 g/l) and then quenched with 600 μL of 80% iced methanol on dry ice. Following 10 min of sonication on slurry ice using a bath sonicator (Bioruptor) with the cycling 30 s on/off at the highest settings, the homogenates were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were collected and supplemented with 750 ng of myristic acid-D₂₇ (an internal standard; Sigma-Aldrich, ON, Canada) and dried overnight in a cold vacuum centrifuge (Labconco). The dried samples were reconstituted with 30 μL of methoxyamine-HCl (10 mg/mL dissolved in pyridine; Sigma) and incubated for 30 min at room temperature. Next, the samples were derivatized with MTBSTFA (Sigma). After incubation for 1 h at 70 °C, 1 μL of each derivatized sample was injected into the GC/MS instrument (5975C, Agilent). Data were acquired in scan mode and analyzed with the MassHunter software (Agilent) as described^{28 (link),80 (link)}. The level of each metabolite was normalized by the intensity of myristic acid-D₂₇ and the average cell number of three independent wells per treatment (run in parallel), on the day of each separate biological repeat experiment.