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Methods Enzymol. 2000;312:32-45.

Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate by tandem mass spectrometry.

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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050, USA.


Free sphingoid bases such as sphingosine, sphinganine, and the respective phosphorylated bases, as well as the complex sphingolipids ceramides, glucosylceramide, and sphingomyelin, all dissociate to form structurally distinctive product ions. For sphingomyelin these ions are characteristic of their phosphorylcholine headgroup and are observed at m/z 184. The other sphingolipids dissociate to form carbocations characteristic of their sphingoid base. For common mammalian sphingoid bases such as d18:1 or d18:0 these product ions are detected at m/z 264 or 266, respectively. However, changes in the sphingoid base chain length, degree of unsaturation, or other modifications may correspondingly result in a shift in m/z of [figure: see text] this product ion. Additionally, the kinetics that govern the formation of these product ions is affected by the presence of a delta 4 double bond. Thus, internal standards for each type of sphingoid base are required for quantitative data. Structurally distinctive product ions, when used with either precursor ion or constant neutral loss scans allow highly specific and sensitive methods for sphingolipid analysis. They serve to greatly reduce background chemical noise, and enhance detection of sphingolipids at very low concentrations. This occurs by allowing only those ions that dissociate to yield a specific product ion or neutral loss to be passed to the detector. Additionally, these scans reveal the exact combinations of headgroup, sphingoid base, and fatty acid in a complex mixture by mass. The free sphingoid bases and Cer readily decompose in the ion source, whereas GlcCer and SM do not. Finally, each individual sphingolipid species fragmented optimally at a different collision energy, precluding the use of either precursor ion or neutral loss scans for quantitation. Multiple reaction monitoring (MRM) experiments directly address the issues regarding accurate quantitation of sphingolipids that precursor ion and neutral loss scans cannot. In these experiments both ionization and dissociation parameters are optimized for each individual species. By detecting only specific precursor and product ion pairs instead of scanning wide m/z ranges maximum sensitivity is attained. Furthermore, relative ion abundance data are not biased with regard to instrumental parameters. At this point simple loop injections can be used with the MRM scanning methods developed to observe changes in sphingolipid type and quantity in crude extracts on a class-by-class basis. This, however, is labor intensive requiring multiple injections and multiple runs for each class in order to obtain a complete picture of all sphingolipids present. As an alternative [figure: see text] to loop injections, HPLC-MS/MS methods are being developed. In these methods sphingolipids are eluted by class, thus, each individually optimized MRM method can be used at specific times in an LC run. This provides a highly sensitive and accurate quantitation as well as a complete picture of all sphingolipids in a single run (Fig. 6). Additionally, this methodology is amenable to automation and can be used for high-throughput screening of multiple samples.

[Indexed for MEDLINE]

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