Innovative methods to advance the analysis of intact glycolipids by mass spectrometry

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Rodell C. Barrientos (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Qibin Zhang

Abstract: The common etiological feature among the majority, if not all, of human diseases, is the impairment of crucial cellular events stemming from complex reactions at the molecular level. Precise understanding of these processes, such as structure-function relationships between biomolecules, could help guide the design of successful clinical interventions to prevent or halt diseases. Consequently, it is important to measure and characterize biomolecules at the finest possible granularity. Glycolipids, biomolecules comprised of a sugar (glycan) head and a lipid tail, serve as one of the key components of the cell membrane. Aberrant glycolipid metabolism is a hallmark of many pathologies, which accentuates their importance in disease pathogenesis. Despite their vital role, our present understanding of glycolipid biology remains incomplete, partly because of the dearth of technologies available to study them. Mass spectrometry (MS) has inarguably fueled much of our current knowledge on glycolipids. But, contemporary methods using this platform are not powerful enough to either detect low abundant glycolipids or elucidate subtle structural details that could facilitate uncovering of their precise biological functions. Often, the glycan and lipid components are analyzed separately, making correlation between structure and biological function impossible. Recent evidences have showcased that both glycan and lipid moieties dictate the overall function of glycolipids, which underlines the importance of structural analysis at the intact molecule level. In this respect, this dissertation was conceived to address the following aims, 1) To develop innovative MS methodologies to measure and characterize glycolipids in its intact, native form; and 2) To apply the developed methods in reasonably complex biological mixtures. Each method developed here is in-line with the following objectives: 1) to enhance detectability of glycolipids by increasing ionization efficiency, 2) to facilitate structural analysis, and 3) to improve quantification of glycolipids with potential for high-throughput applications. Both aims were addressed in four standalone projects that employed both commercially available standard glycolipids and complex biological samples. The first two projects sought to establish a method to determine double bond locations in unsaturated glycolipids. We exploited ozone-induced dissociation MS (OzID-MS), a relatively new fragmentation technique that uses ozone gas inside the collision cell of MS in lieu of conventional inert gas. Ozone reacts selectively to carbon-carbon double bonds, as such, the products of the ozonolysis in situ could be detected by MS and being used to locate the double bonds. Using complex bovine brain sample, this method revealed low abundant glycolipids, mostly isomers and isobars, that are otherwise non-detectable and non-distinguishable when conventional methods are used. We also discovered that the use of different adducts, such as [M+Na]+, [M+Li]+, and [M+H]+, could provide distinct OzID-MS patterns. Thus, to rationalize the observed OzID-MS data, theoretical calculations were performed to establish the structures of ionized glycolipids in the gas-phase. The in silico generated models were consistent with the experimentally observed fragmentation patterns. Overall, these projects emphasized that innovative approaches like OzID-MS could uncover previously unknown molecular species in a complex sample and the use of different adducts could provide distinct levels of structural detail. The third project aimed to establish a method that exploits a relatively new approach to quantitation called isobaric labeling, a technique that has been extensively used in proteomics and glycomics fields as a multiplexed analytical tool. It involves the covalent attachment of a molecular tag to an analyte that generates reporter ions when fragmented in MS. The intensities of the reporter ions serve as surrogate measures of their relative concentration in the sample. Because this tag only reacts with a reactive aldehyde or ketone, which is absent in native glycolipids structures, we first employed a chemoselective oxidation approach to introduce a reactive site in the intact glycolipids, using sialic acid-containing glycolipids called gangliosides as a model. When applied to complex porcine brain total lipids extract, this method not only enabled multiplexed analysis of up to six independent samples and improved sensitivity of gangliosides by two orders, but also provided rich spectra that facilitated the structural analysis of both the sugar head group and lipid backbone. The fourth project addressed the increasing demand for heavy isotope labeled internal standards, which are currently limited and costly, while improving the detectability, facilitating structural characterization, and enabling multiplexed analysis. In this project, we employed permethylation, a reaction that converts active protons in the molecule to a methyl group using methyl iodide. In this so-called differential isotope labeling approach, using methyl iodide with either light (12C) or heavy (13C) carbon isotopes, two different samples were separately labeled, one of which is a pooled aliquot of individual samples, then mixed and analyzed by LC-MS. The pooled sample, labeled with heavy carbon isotope served as a universal internal standard which was spiked to individual samples at constant amount. Samples were analyzed using reversedphase liquid chromatography mass spectrometry (RPLC-MS), and the resulting peak areas were used to calculate the 12C/13C ratio as a surrogate measure of the relative concentration of analytes in each sample. Using an in vitro model of Gaucher’s disease, characterized by accumulation of neutral glycolipids, temporal changes in glycolipid profile were measured and each glycolipid was annotated using retention time and MS/MS fragmentation. [This abstract has been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]

Additional Information

Language: English
Date: 2019
Gangliosides, Glycolipids, Isobaric labeling, Mass spectrometry, Ozone-induced dissociation, Permethylation
Mass spectrometry

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