PL measurements alpha syn KD 2022
Based on the research studies showing an altered PUFAs plasma membrane composition upon alterations to alpha synuclein expression levels, we hypothesised that reducing alpha syn in LUHMES dopaminergic neurons may have altered the plasma membrane composition. Phospholipid molecular species were therefore measured in LUHMES cells NT, Ctrl KD, asyn KD and ACSL4 KD under basal control or 20uM Arachidonic acid (AA) conditions. Here we report a selective and prominent reduction of ether-linked phospholipids (PC and PE) upon alpha syn and ACSL4 depletion. Such reduction in ether-PL may explain the ferroptosis resistance phenotype observed.
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Lipids were extracted from cells by homogenizing 3 million cells with 2mL of NaCl solution in water (0.73%). Lipids were extracted with 10 ml of CHCl3/CH3OH (2:1, v/v), and vortexed for 1 minute. Mixture was centrifuged at 3000 rpm for 3 minutes. The upper phase was discarded and the lower phase collected through a protein interface using a Pasteur pipette. After evaporation, the lipid extract (lower phase) was re-dissolved in 200μL of CHCl3/CH3OH (2:1, v/v) and stored, under nitrogen, at -20°C . In the 200μl lipid extract, 10μl of internal standards mixture containing 320μg/ml PC(14:0/14:0) and 160μg/ml PE(14:0/14:0) were added. The process of identification and quantification of phospholipids species was performed on a Thermo UltiMateTM 3000 coupled to an Orbitrap FusionTM Tribrid Mass Spectrometer equipped with an EASY-MAX NGTM Ion Source (H-ESI) (Thermo Scientific). Separation of phospholipid classes was achieved under HILIC conditions using Kinetex Hilic 100 x 2.1 mm, 1.7μm column (Phenomenex), with a flow of 0.500 mL.min−1. The mobile phase consisted of (A) CH3CN/H2O (96/4, v/v) containing 10 mM ammonium acetate and (B) CH3CN/H2O (50/50, v/v) containing 10 mM ammonium acetate. The injection volume was 10 μL at 50°C. PL species were detected by high resolution mass spectrometryanalysis, and H-ESI source parameters were optimized and set as follows: ion transfer tube temperature of 285°C, vaporizer temperature of 370°C, sheath gas flow rate of 35 au, sweep gas of 1 au and auxiliary gas flow rate of 25 au. Positive and negative ions were monitored alternatively by switching polarity approach with a static spray voltage at 3500V and 2800V in positive and negative respectively. Mass spectra in full scan mode were obtained using the Orbitrap mass analyzer with the normal mass range and a target resolution of 240,000 (FWHM at m/z 200), on a mass range to charge ratio m/z form 200-1600 using a Quadrupole isolation on a normal mass range. All MS data were recorded using a max injection time of 100 ms, automated gain AGC target (%) at 112.5, RF lens (%) at 50 and one microscan. An Intensity Threshold filter of 1.103 counts was applied. For MS/MS analyses, data-dependent mode was used for the characterization of PL species. Precursor isolation was performed in the Quadrupole analyzer with an isolation width of m/z 1.6. Higher-energy Collisional Dissociation was employed for the fragmentation of PL species with optimized stepped collision energy of 27%. The linear ion trap was used to acquire spectra for fragment ions in data-dependent mode. The AGC target was set to 2.104 with a max injection time of 50 ms. All MS and MS/MS data were acquired in the profile mode. The Orbitrap Fusion was controlled by XcaliburTM 4.1 software. Data of high accuracy and the information collected from fragmentation spectra, with the help of the LipidSearchTM software (Thermo) and the LIPID MAPS® database were used for PL species identification.