Neutral GSL and monosialoganglioside mixtures were purchased from Matreya LLC (PA, USA). Chloroform, methanol, and DCE of special reagent grade were purchased from Wako Pure Chemical Industries, Ltd (Tokyo, Japan) for lipid extraction and purification. For LC/MS/MS, ammonium hydrogen carbonate (proteomics grade, Wako Pure Chemical Industries, Ltd, Japan), and LC–MS-grade methanol, 2-propanol, and water (Sigma-Aldrich, USA) were used.
3T3-L1 fibroblasts were differentiated into adipocytes as described previously . Briefly, we cultured the fibroblasts in D-MEM containing 10 % calf serum until subconfluent, replaced the medium with fresh D-MEM containing 10 % FBS, and cultured them further for 3 days. Fibroblasts were differentiated with supplemental IBMX, dexamethasone, troglitazone, and insulin.
Lipid Extraction and DCE Washing
3T3-L1 adipocytes were washed with phosphate-buffered saline (PBS) and harvested into screw-capped centrifugal glass tubes. Total lipids were extracted using chloroform/methanol/PBS, (1:2:0.8, v/v) and chloroform/methanol (C/M) 1:2, C/M 1:1, and C/M 2:1. We dissolved the delipidated residue in 0.5 N NaOH, diluted it five times with water, and conducted protein quantification by the bicinchoninic acid method. DCE washing was performed as described previously . Briefly, we dried total lipids under N2 stream to maximally cover the inner surface of the glass tube by dried lipid. Highly hydrophobic materials among the dried lipids were washed off 3 times with 2 ml of ice-cold DCE by pipetting. In DCE-wash, the resulting residual fraction was subjected to mild alkali hydrolysis, in which phospholipids were broken down and removed, followed by desalination with Sep-pakC18 and TLC.
Samples dissolved in C/M 1:1 were developed on a silica gel HPTLC plate (Merck, Germany) in C/M/0.2 % CaCl2 at 60:40:9 (v/v) and visualized by spraying primuline reagent for general lipid and orcinol–H2SO4 reagent for GSL, or primuline reagent and cupric sulfate reagent.
Semipurified samples dissolved in LC–MS-grade methanol were analyzed on LCMS-IT-TOF quadrupole ion trap time-of-flight mass spectrometer (Shimadzu, Japan) according to Kuwahata et al. .