Vanquish uplc system

One year post-production, eight aliquots that had been stored in LN₂ vapor-phase freezers (−140 to −190 °C) were analyzed using the same extraction method in order to assess stability of the amended compounds. Extracts were analyzed by LC-MS/MS using a Vanquish UPLC system coupled to a Q Exactive Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) high-resolution accurate mass spectrometer. The separation was accomplished with an Agilent XDB-C18 column (Santa Clara, CA, USA) using the same mobile phases and temperature program as previously described. The liquid chromatography effluent was introduced to the mass spectrometer using a heated electrospray ionization (HESI) source operating at 3500 V. The sheath gas was set to 40 au, the auxiliary gas was set to 10 au, the ion transfer tube was held at 250 °C and the vaporizer was held at 300 °C. The MS2 was set at a resolution of 17,500, the quad isolation was set to 4 Da, the normalized HCD collision energy was 60%, the AGC was set to 2.0 × 10⁵ and the maximum injection time was 100 ms. Integration of product ions from the compounds of interest was completed as previously described.

The cells were cultured with [U-¹³C₆] glucose or fructose for a certain period of time, washed twice with ice-cold PBS, and lysed with 80% methanol (in water). Targeted LC-MS analyses were conducted on a Q Exactive™ Orbitrap™ Mass Spectrometer (Thermo Scientific) coupled to a Vanquish UPLC system (Thermo Scientific), and the Mass Spectrometer was operated in polarity-switching mode. A SeQuant® ZIC®-HILIC column (2.1 mm i.d. × 150 mm, Merck) was used for separation of metabolites, and the flow rate was 150 μL/min. Buffers consisted of 100% acetonitrile for A, and 0.1% ammonium hydroxide / 20 mM ammonium acetate in water for B. Gradient ran from 85 to 30% A for 20 min, followed by a wash with 30% A and re-equilibration at 85% A. Metabolites and their ¹³C-isotopologues were identified on the basis of standard retention times and exact mass within 5 ppm. The relative quantification was performed based on the metabolite peak area. All data analyses were performed using in-house written scripts.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based metabolomics was performed on deproteinated plasma collected immediately before and for 5 hours after mixed meal ingestion. Polar metabolites were analyzed by using a Thermo QExactive orbitrap mass spectrometer coupled to a Thermo Vanquish UPLC system, as previously described (Garratt et al., 2018 (link)). Bioactive lipids (e.g. free fatty acids, eicosanoids, and bile acids) were profiled on the same LC-MS/MS system, as previously described (Lagerborg et al., 2019 (link); Watrous et al., 2019 (link)). A total of 216 metabolites were annotated by using an in-house library of commercially available standards or MS/MS fragmentation patterns. Metabolite abundances were log2 transformed for presentation and statistical analysis. Gas chromatography mass spectrometry with isotopically-labeled internal standards was used to measure plasma short-chain fatty acids (Rey et al., 2013 (link)).

Targeted polar metabolomics were performed at the shared resource at Cornell University Medical Center, Metabolomics core on BALF harvested at multiple time points post inoculation and intracellular bacterial metabolites. For intracellular metabolites, bacteria were lysed with multiple freeze thaws cycles with liquid nitrogen and 100% ethanol with dry ice. Metabolites were extracted using 80% methanol. Targeted LC/MS analyses were performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled to a Vanquish UPLC system (Thermo Scientific). The Q Exactive operated in polarity-switching mode. A Sequant ZIC-HILIC column (2.1 mm i.d. × 150 mm, Merck) was used for separation of metabolites. The flow rate was set at 150 μL/min. The buffers consisted of 100% acetonitrile for mobile A, and 0.1% NH₄OH/20 mM CH₃COONH₄ in water for mobile B. The gradient ran from 85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A. Metabolites were identified on the basis of exact mass within 5 ppm and standard retention times. Relative metabolite quantitation was performed based on peak area for each metabolite. PCA component analysis was performed using either MetaboAnalyst or independently generated R script.

Polar metabolomic analysis was performed at the Weill Cornell Medicine (WCM) Meyer Cancer Center Proteomics & Metabolomics Core Facility. Metabolites were extracted from 2×10⁶ cells using 80% methanol. Pre-cooled 80% methanol (1 mL) was added to each sample and kept in −80°C overnight. Samples were then centrifuged at 4°C for 15 minutes at 14,000 rpm. The supernatants were extracted. Targeted LC/MS analyses were performed on a Q Exactive Orbitrap mass spectrometer (Thermo-Fisher) coupled to a Vanquish UPLC system (Thermo-Fisher). The Q Exactive operated in polarity-switching mode. A Sequant ZIC-HILIC column (2.1 mm i.d. × 150 mm, Merck) was used for separation of metabolites. Flow rate was set at 150 μL/min. Buffers consisted of 100% acetonitrile for mobile A, and 0.1% NH₄OH - 20 mM CH₃COONH₄ in water for mobile B. Gradient ran from 85% to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A. Metabolites were identified on the basis of exact mass within 5 ppm and standard retention times. Relative metabolite quantitation was performed based on peak area for each metabolite. All data analysis was done using in-house written scripts.