The lipid extraction (adapted from Matyash et al. 23 (link)) was carried out in high grade polypropylene deep well plates. Fifty microliters of diluted plasma (50×) (equivalent of 1 μL of undiluted plasma) was mixed with 130 μL of ammonium bicarbonate solution and 810 μL of methyl tert-butyl ether/methanol (7:2, v/v) solution was added. Twenty-one microliters of internal standard mixture was pre-mixed with the organic solvents mixture. The internal standard mixture contained: 50 pmol of lysophasphatidylglycerol (LPG) 17:1, 50 pmol of lysophosphatic acid (LPA) 17:0, 500 pmol of phosphatidylcholine (PC) 17:0/17:0, 30 pmol of hexosylceramide (HexCer) 18:1;2/12:0, 50 pmol of phosphatidylserine (PS) 17:0/17:0, 50 pmol of phosphatidylglycerol (PG) 17:0/17:0, 50 pmol of phosphatic acid (PA) 17:0/17:0, 50 pmol of lysophposphatidylinositol (LPI 17:1), 50 pmol of lysophosphatidylserine (LPS) 17:1, 1 nmol cholesterol (Chol) D6, 100 pmol of diacylglycerol (DAG) 17:0/17:0, 50 pmol of triacylglycerol (TAG) 17:0/17:0/17:0, 50 pmol of ceramide (Cer) 18:1;2/17:0, 200 pmol of sphingomyelin (SM) 18:1;2/12:0, 50 pmol of lysophosphatidylcholine (LPC) 12:0, 30 pmol of lysophosphatidylethanolamine (LPE) 17:1, 50 pmol of phosphatidylethanolamine (PE) 17:0/17:0, 100 pmol of cholesterol ester (CE) 20:0, 50 pmol of phosphatidylinositol (PI) 16:0/16:0. The plate was then sealed with a teflon-coated lid, shaken at 4°C for 15 min, and spun down (3000 g, 5 min) to facilitate separation of the liquid phases and clean-up of the upper organic phase. Hundred microliters of the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Dried lipids were re-suspended in 40 μL of 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, v/v/v) and the wells were sealed with an aluminum foil to avoid evaporation and contamination during infusion. All liquid handling steps were performed using Hamilton STARlet robotic platform with the Anti Droplet Control feature for organic solvents pipetting.
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