Characterization of polar lipids using mass spectrometry
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A sensitive, efficient one-step method to separate and define the individual structures of the components in complex mixtures of polar lipids, based on gradient reversed phase high performance liquid chromatography coupled to electrospray ionization (ESI) mass spectrometry (MS), was developed. This online system was applied to both standards and biological samples, including the polar lipids that are apparently tightly bound and therefore extracted with the prion protein. The system generated highly resolved spectra and enabled definitive lipid characterization by ESI-MS/MS. Coupling thin-layer chromatography (TLC) with ESI-MS allowed the acquisition of high resolution mass spectra of acidic glycosphingolipids with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure matrix-assisted laser desorption/ionization MS. Based on observations of oxidation under ambient conditions made during development of the on-line TLC-ESI-MS method, a simple approach was established to elucidate carbon-carbon double bond positions in unsaturated lipids. Lipids were deposited onto various surfaces and the products resulting from their oxidation under standard laboratory conditions were observed by unmodified ESI-MS. It is shown that singlet oxygen acts in parallel with ozone to carry out the oxidation of unsaturated lipids. Direct sampling by TLC-ESI-MS provided a powerful approach to elucidate detailed structural information for biological samples; e.g., for a bovine brain total lipid extract, it was possible to distinguish among the isomers of phosphatidylserine and N,N-dimethyl phosphatidylethanolamine based on their double bond positions. A modified sulfatide extraction method and workflow were designed, using nanoESI in combination with tandem MS methods performed on the L TQ-Orbitrap TM MS, and were employed for identification and comprehensive structural characterization of sulfatides. In addition to 20 sulfatides, more than 100 other lipids were identified in a human milk fraction that showed activity against the human immunodeficiency virus and is thus of interest in the search for agents to protect infants and immunocompromised individuals. In summary, the research on the analysis of polar lipids presented here demonstrates several very effective new methods that facilitated chromatographic separation, improved the effectiveness of on-line MS characterization, and provide a new tool for locating sites of unsaturation in biological lipids.
Thesis (Ph.D.)--Boston University