Thermo qexactive plus

HRESIMS data were collected and in positive and negative ionization modes using a Thermo QExactive Plus mass spectrometer (ThermoFisher) equipped an electrospray ionization (ESI) source and via an Acquity UPLC system (Waters Corp). The higher-energy collisional dissociation (HCD) cell used a normalized collision energy of 30 eV for all the compounds to obtain MS/MS data. The UPLC separation was performed using an Acquity BEH C₁₈ column equilibrated at 40 °C and a flow rate set at 0.3 mL/min. The mobile phase consisted of 15% CH₃CN–H₂O (0.1% formic acid) for 0.5 min, and then a linear gradient from 15% CH₃CN to 100% CH₃CN over 6 min, and 1 min holding 100% CH₃CN before returning to the starting conditions. Samples were dissolved in MS grade methanol and filtered through a 0.2 μm Acrodisc (Waters) filter. Tentative metabolite identification was performed by comparison of HRMS data, UV maxima and fragmentation patterns (MS/MS data) with those contained in the Dictionary of Natural Products [20 ] reference compounds refined for Thevetia plant metabolites.

The mass spectrometer Thermo Q-Exactive Plus (Thermo Scientific, San Jose, CA, USA) was equipped with heated electrospray ionization (HESI) source. Capillary temperature and vaporizer temperature were set at 330 and 280 °C, respectively, while the electrospray voltage was adjusted at 3.50 kV (operating in both positive and negative mode). Sheath and auxiliary gas were 35 and 15 arbitrary units, with an S lens RF level of 60.
The mass spectrometer was controlled by the Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA). The exact masses of the compounds were calculated using Qualbrowser in Xcalibur 3.0 software. The mass scan range was set in the range of m/z 100–1000.
The column was a Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm particle size). The mobile phases were (a) water with 0.1% (v/v) formic acid and (b) methanol with 0.1% (v/v) formic acid. The optimized elution conditions were as follows: holding at 10% B for 2 min, a linear gradient from 10% to 13% B (all v/v) (2 to 4 min), 13% to 15% B (4 to 10 min), 15% to 17% B (10 to 15 min), 17% to 21% B (15 to 19 min), 21% to 29% B (19 to 24 min), 29% to 53% B (24 to 29 min), 53% to 75% B (29 to 35 min), 75% to 100% B (35 to 36 min), isocratic 100% B for 1 min, and then back to 7% B in 1 min. The flow rate was 0.3 mL/min. The column temperature was 35 °C. The injection volume was 2 μL.

For stable isotope labeling, MCEC were incubated in DMEM containing 10 mM glucose and 2 mM glutamine with either 1 mM uniformly labeled sodium D‐[¹³C₄]‐β‐hydroxybutyrate or 1 mM uniformly labeled [¹³C₄]‐acetoacetate for 24 h. Samples were collected in methanol and extracted and analyzed as previously described (Puchalska et al, 2019 (link)). In brief, isotope‐labeled samples were extracted by performing three cycles of vortexing, freeze‐thawing and sonication. The protein‐free samples were analyzed by coupled liquid chromatography‐mass spectrometry. Liquid chromatography was performed using a Vanquish Horizon system (Thermo Fisher Scientific) with a Luna NH2 column (Phenomenex) with hydrophilic interaction liquid chromatography and mass spectrometry was performed on a Thermo Q Exactive Plus (Thermo Fisher Scientific) operated in negative mode. Data were processed using the Compound Discoverer 3.0 software. Lipid‐species identities are putative. TCA cycle intermediates, amino acids, and UDP species are identified by use of standards and MS/MS fragmentation pattern matching to open source databases.

Metabolite spectra were generated using a Thermo Vanquish LC connected to a Thermo Q-Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA, USA). Pure chemical standards were dissolved in appropriate solvent, and loop injections into the mass spectrometer were performed, where we acquired fragmentation spectra at a variety of normalized collision energies. Libraries were created by summing the spectra from the three relevant energies (20, 40, and 80 NCE settings in positive mode, and 20, 50, and 100 NCE in negative mode) to match the data acquisition, using stepped collision energies. We have made these libraries publicly available as msp files (Dataset S2).