4000qtrap mass spectrometer

Triacylglycerol (TAG), free fatty acid (FFA), and cholesterol in mouse kidney tissues were analyzed with mass spectrometry-based lipidomics. Briefly, the mouse kidney samples were homogenized in water (1:4, w/v) using Omni Bead Ruptor. A modified Bligh and Dyer method was used to extract TAG, FFA, and cholesterol from 50 μL of homogenate. Internal standards of TAG (Nu-Chek Prep #T404), FFA (Cambridge Isotopes #DLM-2893–0.5) and cholesterol (Toronto Research Company #C432503) were added to the samples before extraction. The FFA was derivatized with 4-aminomethylphenylpyridium to improve mass spectrometric sensitivity. Measurement of TAG, FFA and cholesterol was performed with a Shimadzu 20AD HPLC system coupled to a 4000QTRAP mass spectrometer operated in positive multiple reaction monitoring mode. The electrospray ionization was used for TAG and FFA, and air pressure chemical ionization was used for cholesterol. Data processing was conducted with Analyst 1.6.3. Quality control (QC) samples were prepared by pooling the aliquots of the study samples and were used to monitor the instrument stability. Only the lipid species with CV < 15% in QC sample were reported. The data were reported as the peak area ratios of the analytes to the corresponding internal standards.

DKO and TKO B-ALL cells were plated at 500,000 cells/mL in regular or low glucose media. After 24 hours, 3 million cells per sample were centrifuged at 1,000 rpm for 5 minutes and washed once with 1x PBS. Supernatants were removed and cell pellets were shipped on dry ice to the Metabolomics Facility at Washington University (P30 DK020579) for analysis. Cell suspensions (1 × 10⁷ cells/mL) were prepared by vortexing cell pellets with H₂O. Protein precipitation was performed to extract free fatty acids (FFA) from 50 μL of cell suspension. The d4-FFA (16:0) was used as an internal standard, which was added to the samples before extraction. Analysis of FFAs was performed in positive MRM mode on a 4000QTRAP mass spectrometer coupled to a Shimadzu 20A HPLC system. Data processing was conducted with Analyst 1.6.3. Quality control (QC) samples were prepared by pooling aliquots of the samples and were used to monitor instrument performance. The QC samples were injected between every five experimental samples. Only the lipid species with coefficient of variance less than 15% in QC injections were reported. The relative quantification of lipids was provided as the peak area ratios of the analytes to the corresponding internal standards.