DOI: 10.5281/zenodo.5613050
title: LC-MS raw data_Lysophosphatidic Acid Shifts Metabolic and Transcriptional Landscapes to Induce a Distinct Cellular State in Human Pluripotent Stem Cells

description: <strong>LC-MS/MS analysis</strong> <strong>Metabolite extraction</strong> For LC-MS/MS quantification, cell sample preparation was conducted as described in the previous literatures (Ying, Kimmelman et al. 2012, Zhang, Badur et al. 2016). Briefly, the spent medium was removed, and cells were rinsed with 1 mL/well 0.9% (w/v) saline twice. Then 0.5 mL/well -80°C 0.2 μg/mL norvaline containing 80% methanol was added to quench the metabolism. Cells were scraped off into 1.5-mL eppendorf tube and stored in -80℃ overnight. The mixtures were vortexed and then centrifuged 12500 × <em>g </em>for 15 min at 4℃. The supernatant was used for LC-MS analysis. <strong>LC-MS/MS method</strong> Waters Xevo TQD coupled with Waters Acquity UPLC system was used for quantification. Acquity UPLC BEH HILIC column (2.1 × 100 mm, 1.7 μm), Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), and Acquity UPLC BEH amide column (2.1 × 100 mm, 1.7 μm) were used for the separation of metabolites. Column temperature was set at 40 °C. For the quantification of norvaline, amino acids, GSH, GSSG, SAH, SAM, ascorbic acid and myo-inositol, amide column was used for the separation. Acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B) were used as mobile phases. The gradient setting is: 0-4 min, 99% A to 90% A; 4-10 min, 90% A to 67% A; 10-13 min, 67% A to 1% A; 13-15 min, 1% A; 15-16.5 min, 1% A to 99% A; 16.5-20 min, 99% A. Flowrate was set as 0.4 mL/min. For the quantification of metabolites involved in TCA cycle, energy related and ribonucleotides, an amide column was used for the separation. Acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B) were used as mobile phases. The gradient setting is: 0-2 min, 80% A; 2-3 min, 80% A to 20% A; 3-5 min, 20% A; 5-6 min, 20% A to 80% A; 6-10 min, 80% A. Flowrate was set as 0.4 mL/min. For the quantification of acetate, acetyl-CoA and metabolites involved in glycolysis and pentose phosphate pathway, HILIC column was used for the separation. Acetonitrile (A) and 10 mM ammonium bicarbonate were used as mobile phases. The gradient setting is: 0-2 min, 10% A; 2-5 min, 10% A to 5% A; 5-6 min, 5% A to 10% A; 6-10 min, 10% A. Flowrate was set as 0.2 mL/min. For the quantification of LPA, LPC and PC, HILIC column was used for the separation. Acetonitrile (A) and 10 mM ammonium bicarbonate aqueous solution (B) were used as mobile phases. The gradient setting is: 0-2 min, 95% A; 2-4 min, 95% A to 10% A; 4-7 min, 10% A; 7-9 min, 10% A to 95% A; 9-15 min, 95% A. Flowrate was set as 0.2 mL/min. For the quantification of CDL lipids, C18 column was used for the separation. 98% Acetonitrile aqueous solution (A) and 10 mM ammonium acetate 90% acetonitrile aqueous solution (B) were used as mobile phases. The gradient setting is: 0-5 min, 0.1% A; 5-6 min, 0.1% A to 99.9% A; 6-11 min, 99.9% A; 11-12 min, 99.9% A to 0.1% A; 12-15 min, 0.1% A. Flowrate was set as 0.4 mL/min. Argon was used as source gas, capillary voltage was 3500 V, and desolvation temperature was 500 °C. Multiple reaction monitoring (MRM) was conducted, and the ion transitions are listed in the supplemental Table S2. Selected ion recording (SIR) was conducted for the detection of CDL-related lipids, and the setting is listed in the supplemental Table S3. Standard solutions of TCA metabolites (100 μg/mL) and intermediates of glycolysis and pentose phosphate pathway (10 μg/mL) were prepared to confirm the retention time. Peak intensity of product ion was used for the quantification. Data analysis was performed by TargetLynx software (Waters) with statistical analysis in Graphpad Prism (version 8.4.0) and R.
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