Metabolomic Profiling of CD4+ T cells into Tfh Cells
Description
This dataset comprises metabolomic profiling of CD34 lineage cell differentiation into Tfh cells in transplant arteriosclerosis. Metabolic signatures were analyzed using ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Both untargeted and targeted metabolomics approaches were employed to assess metabolic flux in CD4+ T cells at different differentiation stages, with specific focus on mitochondrial one-carbon metabolism. CD4+ T cells were isolated from C57BL/6J background mice lymphoid tissues , and metabolite extraction was performed using 80% methanol, followed by centrifugation and filtration. The LC-MS analysis was conducted in both positive and negative ionization modes, and metabolite identification was performed based on MS1 and MS2 spectra using in-house and public databases. Data processing included peak alignment and normalization using internal standards.
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In terms of the items of contract, samples were analyzed based on UHPLC-HRMS/MS -based non-targeted metabolomics platform. Valine-13C5-15N and leucine-13C6 were purchased from Sigma-Aldrich (Shanghai, China). Phenylalanine-d5, 3-chloro-D-phenylalanine, octanoic-d15 acid, decanoic-d19 acid, octadecanoic-d35 acid, tetradecanoic-d27 acid, hexadecanoyl-L-carnitine-d3 and decanoyl-L-carnitine-d3 were obtained from C/D/N Isotopes (Pointe-Claire, Quebec, Canada). The above compounds were utilized as internal standards for data normalization of metabolomics. The reference compounds for identification were mainly purchased from Sigma-Aldrich (Shanghai, China), Santa Cruz Biotechnology (Shanghai, China), Toronto Research Chemicals (Toronto, ON, Canada), and Aladdin Bio-Chem Technology (Shanghai, China). LC-MS grade acetonitrile, methanol, and formic acid were the products of Fisher Scientific. The key instruments include (1) Ultra-High Performance Liquid Chromatography (UHPLC) – High Resolution Tandem Mass Spectrometry (HRMS/MS): ThermoFisher Ultimate 3000 UHPLC and ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE); and (2) High-speed refrigerated centrifuge: Eppendorf 5430R (Hamburg, GER). Cell samples collected in 80% methanol were ultrasonicated for 5 min in ice-water bath and then stand at -40 ° C for 30 min, the mixture was centrifuged at 15000 g and 4 ℃ for 15 min. All supernatant was dried and reconstituted in 50 μL of 50% acetonitrile (containing internal standards) prior to perform UHPLC-HRMS/MS analysis. Quality control (QC) sample was obtained by isometrically pooling all the prepared samples. Chromatographic separation was performed on a ThermoFisher Ultimate 3000 UHPLC system with a Waters ACQUITY UPLC BEH Amide column (2.1mm × 100 mm, 1.7μm). The mobile phases consisted of (A) water with 15mM ammonium acetate and 0.2% ammonium hydroxide and (B) 90% acetonitrile. A linear gradient elution was performed with the following program: 0 min, 90%B; 4 min, 85%B; 11 min, 75%B and held to 18 min; 18.1min, 90%B and held to 20 min. The flow rate was 0.25 mL/min. The extracts were analyzed on a ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry (QE) in Heated Electrospray Ionization Positive (HESI+) and Negative (HESI-) mode, respectively. Spray voltage was set to 3.5 kV for HESI+ and HESI-. Both Capillary and Aux Gas Temperature were 350 °C. Sheath gas flow rate was 40 (Arb). Aux gas flow rate was 10 (Arb). S-Lens RF Level was 50 (Arb). The full scan was operated at a high-resolution of 35000 FWHM (m/z=200) at a range of 70 - 1050 m/z with AGC Target setting at 3×106. Simultaneously, the fragment ions information of top 8 precursors each scan was acquired by Data-dependant acquisition (DDA) with HCD energy at 15, 30 and 45 eV, mass resolution of 17500 FWHM, and AGC Target of 2×105.