Lipid extraction for mass spectrometry lipidomics

Mass spectrometry-based lipid analysis was performed by Lipo-type GmbH (Dresden, Germany) as described (Surma et al., 2021). If not indicated otherwise, 500 μg of tissue were used per extraction. Lipids were extracted using a two-step chloroform/methanol procedure using homogenized tissues re-suspended in a water phase, extracted with chloroform/methanol 10:1 (V:V) and 2:1 (V:V) in the first and second step, respectively (Ejsing et al., 2009). Samples were spiked with internal lipid standard mixture containing: cardiolipin 16:1/15:0/15:0/15:0 (CL, 50 pmol per extraction), ceramide 18:1; 2/17:0 (Cer, 30 pmol), diacylglycerol 17:0/17:0 (DAG, 100 pmol), hexosylceramide 18:1; 2/12:0 (HexCer, 30 pmol), lyso-phosphatidate 17:0 (LPA, 30 pmol), lyso-phosphatidylcholine 12:0 (LPC, 50 pmol), lyso-phosphatidylethanolamine 17:1 (LPE, 30 pmol), lyso-phosphatidylglycerol 17:1 (LPG, 30 pmol), lyso-phosphatidylinositol 17:1 (LPI, 20 pmol), lyso-phosphatidylserine 17:1 (LPS, 30 pmol), phosphatidate 17:0/17:0 (PA, 50 pmol), phosphatidylcholine 17:0/17:0 (PC, 150 pmol), phosphatidylethanolamine 17:0/17:0 (PE, 75 pmol), phosphatidylglycerol 17:0/17:0 (PG, 50 pmol), phosphatidylinositol 16:0/16:0 (PI, 50 pmol), phosphatidylserine 17:0/17:0 (PS, 100 pmol), cholesterol ester 20:0 (CE, 100 pmol), sphingomyelin 18:1; 2/12:0; 0 (SM, 50 pmol), triacylglycerol 17:0/17:0/17:0 (TAG, 75 pmol), Gb3 18:1; 2/17:0; 0 (200 pmol), GM1-D3 18:1; 2/18:0; 0 (200 pmol), GM3-D3 18:1; 2/18:0; 0 (50 pmol), LacCer d18:1/12:0 (50 pmol), Sulfatide d18:1; 2/12:0; 0 (20 pmol) and cholesterol D6 (Chol, 300 pmol). After extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. 1st step dry extract was re-suspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, V:V:V) and 2nd step dry extract in 33% ethanol solution of methylamine in chloroform/methanol (0.003:5:1; V:V:V). All liquid handling steps were performed using Hamilton Robotics STARlet robotic platform with the Anti Droplet Control feature for organic solvents pipetting.