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Journal of Lipid Research, Vol. 46, 163-175, January 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics

Xianlin Han¹ and Hua Cheng

Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110

¹ To whom correspondence should be addressed. e-mail: xianlin{at}wustl.edu

By using shotgun lipidomics based on the separation of lipid classes in the electrospray ion source (intrasource separation) and two-dimensional (2D) MS techniques (Han, X., and R. W. Gross. 2004. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. First published on June 18, 2004; doi: 10.1002/mas.20023, In press), individual molecular species of most major and many minor lipid classes can be quantitated directly from biological lipid extracts. Herein, we extended shotgun lipidomics to the characterization and quantitation of cerebroside molecular species in biological samples. By exploiting the differential fragmentation patterns of chlorine adducts using electrospray ionization (ESI) tandem mass spectrometry, hydroxy and nonhydroxy cerebroside species are readily identified. The hexose (either galactose or glucose) moiety of a cerebroside species can be distinguished by examination of the peak intensity ratio of its product ions at m/z 179 and 89 (i.e., 0.74 ± 0.10 and 4.8 ± 0.7 for galactose- and glucose-containing cerebroside species, respectively). Quantitation of cerebroside molecular species (as little as 10 fmol) from chloroform extracts of brain tissue samples was directly conducted by 2D ESI/MS after correction for differences in ¹³C-isotopomer intensities.

This method was demonstrated to have a greater than 1,000-fold linear dynamic range in the low concentration region; therefore, it should have a wide range of applications in studies of the cellular sphingolipid lipidome.

Abbreviations: 2D, two-dimensional; ESI, electrospray ionization; GalCer, galactosylceramide; GlcCer, glucosylceramide; MS/MS, tandem mass spectrometry; N18:0-d35, perdeuterated N-octadecanoyl; NL, neutral loss; Nm:n, the acyl amide chain of cerebroside molecular species containing m carbons and n double bonds; 2OH, 2-hydroxy

Supplementary key words electrospray ionization • galactosylceramide • glucosylceramide • intrasource separation • sphingolipids • spinal cord • two-dimensional mass spectrometry

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