Lipid composition of eluate from CD1a protein. (a) Chloroform and methanol eluents were subject to electrospray ionization-MS in positive mode. Detected compounds: Phosphatidylcholine (PC), Sphingomyelin (SM), Triacylglycerol (TAG), Phosphatidylinositol (PI), Trihexosylceramide (TriHexCer), Monosialoganglioside (GM3), Globotetrahexosylceramide (Gb4) (b) Lipid identifications are summarized with key identifying fragments shown. Full CID-MS spectra are shown in and . (c) CD1a proteins were extracted in chloroform and methanol, loaded into a silica-diol column and eluted in the normal phase. Individual events are assigned when signal is detected in two or more replicate runs reproducibly detected within mass (20 ppm) and time (30 sec) windows. Retention time of all events (1,336) is divided into three retention time ranges that include lipids with low (n=298), intermediate (n=414) and high (n=624) polarity, shown in comparison to known benchmark lipids, diacylglycerol (DAG), PI, phosphatidyl ethanolamine (PE) and sphingomyelin (SM). (d) Purified lipids or CD1a eluates were loaded onto plate-bound CD1a proteins that had been previously treated with acidic citrate buffer and tested for activation of BC2. Ceramide (Cer), Glucosylceramide (GluCer), Lactosylceramide (LacCer), Globotriaosylceramide (Gb3), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA). Results are representative of at least 3 experiments, and depicted as the mean of triplicate measurements ± standard deviation.