Fatty acid desaturation controls cellular fate decisions between senescence and ferroptosis

Description

Biocrates targeted metabolite profiles for fatty acids and aqueous metabolites from senescent and contol IMR-90 fibroblasts.

Steps to reproduce

Senescent cells (SEN IR) were generated by exposing cells to 10 Gy of ionizing radiation, with sham irradiation non-senescent (NS) serving as controls. Seven days after, BSA or DGLA was administered for 48 h, followed by extraction and analysis as described below. Lipids were isolated and quantitated using a Biocrates MxP Quant 500 kit. Briefly, 20 µL samples were injected into an Agilent 1290 liquid chromatograph using the chromatographic conditions prescribed by the kit. The eluant from the HPLC was connected to an Agilent 6550 iFunnel quadrupole time-of-flight/mass spectrometry (QTOF) mass spectrometer with a Dual AJS electrospray ion source in positive ionization mode. Analyses were performed using the following parameters: gas temperature (TEM) 200°C; drying gas, 13 psi; Vcap, 5500V; nebulizer, 60 psi; nozzle voltage 1000 volts; sheath gas temp 250°C; sheath gas flow 11 L/min; and a reference mass of 149.02332 m/z. MS1 was acquired in the positive ion scanning mode for a mass range of 75-3200 m/z. Quantitation of aqueous metabolites by ultra high performance liquid chromatography (UHPLC) QTOF-MS. Samples were injected into an Agilent 1290 liquid chromatograph equipped with a Biocrates MxP Quant 500 column system. Conditions followed the kit’s instructions. The eluant from the HPLC was connected to an Agilent 6550 iFunnel quadrupole time-of-flight/mass spectrometry (QTOF) mass spectrometer with a Dual AJS electrospray ion source. Analyses were performed using the following ionization parameters: gas temperature (TEM) 225°C; drying gas, 13 psi; Vcap, 3500V; nebulizer, 35 psi; nozzle voltage 50 volts; sheath gas temp 350°C; sheath gas flow 12 L/min; and a reference mass of 84.9595. MS1 acquisition was operated in the positive ion scanning mode for a mass range of 70-1500 m/z. In both cases, data were collected in high-resolution scan mode not in multiple reaction monitoring (MRM) mode as in the kit, relying on the resolving power of the TOF not MS/MS for detection and identification of the compounds. We developed a data processing protocol using Agilent MassHunter Quantitative Analysis. We quantified these compounds using the Biocrates internal standards and calibrations curves. The Agilent Quantitative Analysis software did peak picking, integration, calibration curves and quantitative calculations automatically for most compounds, but some manual integration was required for low level metabolites. The QTOF collected quantitative targeted and untargeted metabolic profiles simultaneously. Because we were not using MS/MS, the isomers of the individual DG’s and TG’s appear as one peak and could not be differentiated. The QTOF allows identification of molecular formulas. Names of DGs and TGs were changed to include the molecular formulas. For example TG(14:0_32:2) in the Biocrates database is TG(C47H82O6) in our database. Peak areas and standard-derived were normalized to total protein.

Institutions

• Jean Mayer Human Nutrition Research Center on Aging

  Massachusetts, Boston

Categories

Funders

• American Federation for Aging Research

  New York, New York

  Grant ID: Grants for Junior Faculty
• Agricultural Research Service

  United States Department of Agriculture

  District of Columbia, Washington

  Grant ID: 58-8050-9-004

Licence