| Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids influence their biological activity, there is a need for comprehensive methods for quantitation of as many individual subspecies as possible. This dissertation describes methods that have been developed and validated for the extraction, liquid chromatographic separation, identification and quantitation of sphingolipids by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail developed by the LIPID MAPS Consortium. The compounds that can be readily analyzed are sphingoid bases and sphingoid base 1-phosphates, as well as more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, and mono- and di-hexosylceramides. For broader utility, the methods have been optimized for two categories of tandem mass spectrometers, a triple quadrupole mass spectrometer and a quadrupole linear-ion trap mass spectrometer. With minor modifications, these methods can be applied to the analysis of isomers such as glucosylceramide and galactosylceramide, and with the availability of additional internal standards, more complex species such as sulfatides can also be quantified. Using these methods 46 species of these compounds have been quantified in RAW264.7 cells, a macrophage cell line.;Quantitation of individual sphingolipid metabolites is possible using liquid chromatography, tandem mass spectrometry, and stable isotope labeling with [13C]palmitic acid can be used to differentiate between metabolites produced by de novo synthesis versus turnover. This approach is more accurate when one knows the isotope enrichment of the precursor pool (in this case, [13C]-palmitoyl-CoA); therefore this dissertation describes methods to analyze both the various isotopic forms of palmitoyl-CoA and sphingolipids through sphingomyelins and monohexosylceramides using two cell models, HEK293 cells and RAW264.7 cells treated with Kdo2-Lipid A. The analysis of the fatty acyl-CoA’s followed a fragmentation mechanism involving a neutral loss of 507 Da (with the charge residing on the acyl-chain and thioester portion of the CoA). The sphingolipid analysis was simplified by the fragmentation of most of the metabolites (sphingoid bases and 1-phosphates, ceramides, and monohexosylceramides) to backbone product ions. For example the presence of the isotopic label in the long chain base, N-acyl linked fatty acid, or both was determined via, m/z 264 for [12 C]sphingosine (d18:1) and m/z 280 for [13 C]sphingosine (m+16, d18:1), versus the m/z of the isotopically labeled precursor, (m+16 versus m+32). For sphingomyelins, which poorly fragment to the individual backbone moieties, the isotope label can be determined by first treating the samples with phospholipase D to produce ceramide 1-phosphates that give the backbone fragments. Sphingolipid isotope metabolic precursor labeling experiments (SIMPLE) give a more accurate estimate of the amounts of sphingolipids made from de novo biosynthesis than is possible without isotopic labeling, or with use of stable isotopes without estimation of the specific activity of the precursor pool.;The alteration of these existing methods for application to collaborative projects is a required task for practical implementation. Several collaborative scenarios are explored and the alterations to standard methodologies are presented. |
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