Comprehensive Characterization of Bovine Milk Lipids: Phospholipids, Sphingolipids, Glycolipids, and Ceramides

A comprehensive survey on phospholipids (PLs), sphingolipids, glycolipids, and ceramides (Cer) of bovine milk was conducted by liquid chromatography tandem-mass spectrometry. A total of 514 species belonging to 15 classes were identified by an automated search tool (LipidSearch) and a manual analysis of MS2 spectra. Over 60 species were detected for the classes of phosphatidylcholine (PC), phosphatidylinositol, phosphatidylserine, and sphingomyelin (SM), between 20 and 45 species for phosphatidylethanolamine (PE), phosphatidic acid (PA), and lactosylceramide (LacCer), and between 7 and 20 species for phosphatidylglycerol (PG), lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, glucosylceramide (GluCer), PC-plasmalogen (PC-P), PE-plasmalogen, ganglioside, and Cer. The structural assignment for PL classes is straightforward based on class-specific product ion/neutral losses and fatty acid-related product ions. By contrast, structural determination of lipid classes containing a backbone of sphingoid bases (SM, GluCer, LacCer, ganglioside, and Cer) is much more challenging, and combining MS2/MS3 information acquired in both positive and negative modes is sometimes needed. The quantification of all 15 classes of lipids was performed at the species level. A large variation in abundance was observed across the lipid classes; PC, PE, and SM are the most abundant polar lipid classes, with concentrations reaching 120, 150, and 90 μmol/L, respectively, whereas PA, PG, and PC-P are present at very low levels (<1.5 μmol/L) and the remaining classes at an intermediate level (2-45 μmol/L). This is the first comprehensive characterization of minor lipid classes from bovine milk and the information provided can be used as a reference for studying milk lipids.