Glycoconjugate Journal

, Volume 28, Issue 2, pp 67–87 | Cite as

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

  • Kouji Tanaka
  • Masaki Yamada
  • Keiko Tamiya-Koizumi
  • Reiji Kannagi
  • Toshifumi Aoyama
  • Atsushi Hara
  • Mamoru Kyogashima


Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated 3,5A ions, indicating Hex1-4Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.


Ceramides Hydroxy-ceramides Nona-octadeca ceramides Neutral glycosphingolipids MALDI-TOF MS High-energy collision-induced dissociation 

Supplementary material

10719_2011_9325_MOESM1_ESM.pdf (38 kb)
Supplemental Fig. I Fragmentation schemes for Fig.2e (d18:1-C24:0), Fig.2f (d18:0-C24:1) and Fig.2g(t18:0-C18:0). (PDF 37 kb)
10719_2011_9325_MOESM2_ESM.pdf (34 kb)
Supplemental Fig. II Fragmentation schemes for Fig.3a (d18:1-C24:0) and Fig.3b (d18:0-C24:1). (PDF 34 kb)
10719_2011_9325_MOESM3_ESM.pdf (48 kb)
Supplemental Fig. III Fragmentation schemes for Fig.3c (d18:1-C18:0h) , Fig.3d (t18:0-C18:0) and Fig.3e (t18:0C24:0h and t20:0-C22:0h). (PDF 48 kb)
10719_2011_9325_MOESM4_ESM.pdf (37 kb)
Supplemental Fig. IV Fragmentation schemes for Fig.6a , HexCer (GalCer d18:1-C24:1) and Fig.6b HexCer (GalCer d18:0-C18:0h) (PDF 37 kb)
10719_2011_9325_MOESM5_ESM.pdf (35 kb)
Supplemental Fig. V Upper part. Compositional comparisons between LacCer from Gb3Cer digested with α-galactosidase and the untreated Gb3Cer, and between Gb3Cer from Forssman glycolipids sequentially digested with α-N-acetylgalactosaminidase and β-N-acetyl-hexosaminidase, and untreated Forssman glycolipid. The asterisks seen in Fig.11 were omitted. Lower part Thin-layer chromatogram of Gb3Cer (equine kidneys, left lane), Forrsman glycolipid (equine kidneys, middle lane) and standard (right lane) developed with chloroform: methanol: water, 65:25:4 (by volume). Bands were visualized with orcinol. (PDF 34 kb)


  1. 1.
    Hirabayashi, Y., Igarashi, Y., Merrill Jr., A.H.: Sphingolipids synthesis, transport and cellular signaling. In: Hirabayashi, Y., et al. (eds.) Sphingolipid Biology, pp. 3–22. Springer, Tokyo (2006)CrossRefGoogle Scholar
  2. 2.
    Hannun, Y.A., Obeid, L.M.: Principles of bioactive lipid signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 9, 139–150 (2008)PubMedCrossRefGoogle Scholar
  3. 3.
    Regina Todeschini, A., Hakomori, S.I.: Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains. Biochim. Biophys. Acta. 1780, 421–433 (2008)PubMedGoogle Scholar
  4. 4.
    Yu, R.K., Yanagisawa, M.: Glycosignaling in neural stem cells: involvement of glycoconjugates in signal transduction modulating the neural stem cell fate. J Neurochem. 103(Suppl 1), 39–46 (2007)PubMedCrossRefGoogle Scholar
  5. 5.
    Masukawa, Y., Narita, H., Shimizu, E., Kondo, N., Sugai, Y., Oba, T., Homma, R., Ishikawa, J., Takagi, Y., Kitahara, T., Takema, Y., Kita, K.: Characterization of overall ceramide species in human stratum corneum. J. Lipid Res. 49, 1466–1476 (2008)PubMedCrossRefGoogle Scholar
  6. 6.
    Kyogashima, M., Tadano-Aritomi, K., Aoyama, T., Yusa, A., Goto, Y., Tamiya-Koizumi, K., Ito, H., Murate, T., Kannagi, R., Hara, A.: Chemical and apoptotic properties of hydroxy-ceramides containing long-chain bases with unusual alkyl chain lengths. J Biochem. 144, 95–106 (2008)PubMedCrossRefGoogle Scholar
  7. 7.
    Panasiewicz, M., Domek, H., Hoser, G., Kawalec, M., Pacuszka, T.: Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells. Biochemistry 42, 6608–6619 (2003)PubMedCrossRefGoogle Scholar
  8. 8.
    Panasiewicz, M., Domek, H., Hoser, G., Fedoryszak, N., Kawalec, M., Pacuszka, T.: The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100. Cell Mol Biol Lett. 14, 175–189 (2009)PubMedCrossRefGoogle Scholar
  9. 9.
    Buschard, K., Blomqvist, M., Månsson, J.E., Fredman, P., Juhl, K., Gromada, J.: C16:0 sulfatide inhibits insulin secretion in rat beta-cells by reducing the sensitivity of KATP channels to ATP inhibition. Diabetes 55, 2826–2834 (2006)PubMedCrossRefGoogle Scholar
  10. 10.
    Iwabuchi, K., Prinetti, A., Sonnino, S., Mauri, L., Kobayashi, T., Ishii, K., Kaga, N., Murayama, K., Kurihara, H., Nakayama, H., Yoshizaki, F., Takamori, K., Ogawa, H., Nagaoka, I.: Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils. Glycoconj J. 25, 357–374 (2008)PubMedCrossRefGoogle Scholar
  11. 11.
    Mahfoud, R., Manis, A., Lingwood, C.A.: Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes. J. Lipid Res. 50, 1744–1755 (2009)PubMedCrossRefGoogle Scholar
  12. 12.
    Hsu, F.F., Turk, J.: Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization. J. Am. Soc. Mass Spectrom. 13, 558–570 (2002)PubMedCrossRefGoogle Scholar
  13. 13.
    Gu, M., Kerwin, J.L., Watts, J.D., Aebersold, R.: Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry. Anal. Biochem. 244, 347–356 (1997)PubMedCrossRefGoogle Scholar
  14. 14.
    Bielawski, J., Pierce, J.S., Snider, J., Rembiesa, B., Szulc, Z.M., Bielawska, A.: Comprehensive quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods Mol. Biol. 579, 443–467 (2009)PubMedCrossRefGoogle Scholar
  15. 15.
    Ikeda, K., Shimizu, T., Taguchi, R.: Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring. J. Lipid Res. 49, 2678–2689 (2008)PubMedCrossRefGoogle Scholar
  16. 16.
    Haynes, C.A., Allegood, J.C., Park, H., Sullards, M.C.: Sphingolipidomics: methods for the comprehensive analysis of sphingolipids. J. Chromatogr. B 877, 2696–2708 (2009)CrossRefGoogle Scholar
  17. 17.
    Scherer, M., Leuthaeuser-Jaschinski, K., Ecker, J., Schmitz, G., Liebisch, G.: A rapid and quantitative LC-MS/MS method to profile sphingolipids. J. Lipid Res. 51, 2001–2011 (2010)PubMedCrossRefGoogle Scholar
  18. 18.
    Levery, S.B.: Glycosphingolipid structural analysis and glycosphingolipidomics. Methods Enzymol. 405, 300–369 (2005)PubMedCrossRefGoogle Scholar
  19. 19.
    Kyogashima, M., Tamiya-Koizumi, K., Ehara, T., Li, G., Hu, R., Hara, A., Aoyama, T., Kannagi, R.: Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS. Glycobiology 16, 719–728 (2006)PubMedCrossRefGoogle Scholar
  20. 20.
    Nakamura, K., Suzuki, Y., Goto-Inoue, N., Yoshida-Noro, C., Suzuki, A.: Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS. Anal. Chem. 78, 5736–5743 (2006)PubMedCrossRefGoogle Scholar
  21. 21.
    Ohta, M., Matsuura, F., Henderson, G., Laine, R.A.: Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus). J. Lipid Res. 48, 656–664 (2007)PubMedCrossRefGoogle Scholar
  22. 22.
    Parry, S., Ledger, V., Tissot, B., Haslam, S.M., Scott, J., Morris, H.R., Dell, A.: Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice. Glycobiology 17, 646–654 (2007)PubMedCrossRefGoogle Scholar
  23. 23.
    Nagahori, N., Abe, M., Nishimura, S.: Structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle. Biochemistry 48, 583–594 (2009)PubMedCrossRefGoogle Scholar
  24. 24.
    Hara, A., Taketomi, T.: Long chain base and fatty acid compositions of equine kidney sphingolipids. J. Biochem. 78, 527–536 (1975)PubMedGoogle Scholar
  25. 25.
    Rumsby, M.G.: A modified column chromatographic method for the recovery of the glycerogalactolipid fraction of nerve tissue. Some observations on the fractionation of nerve tissue glycolipids on silicic acid with chloroform and acetone mixtures. J. Chromatogr. 42, 237–247 (1969)PubMedCrossRefGoogle Scholar
  26. 26.
    Tomita, M., Taguchi, R., Ikezawa, H.: Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus. Biochim. Biophys. Acta. 704, 90–99 (1982)PubMedCrossRefGoogle Scholar
  27. 27.
    Saito, T., Hakomori, S.I.: Quantitative isolation of total glycosphingolipids from animal cells. J. Lipid Res. 12, 257–259 (1971)PubMedGoogle Scholar
  28. 28.
    Hara, A., Kitazawa, N., Taketomi, T.: Abnormalities of glycosphingolipids in mucopolysaccharidosis type III B. J. Lipid Res. 25, 175–184 (1984)PubMedGoogle Scholar
  29. 29.
    Courtois, J.E., Petek, F.: α-Galatosidase from coffee beans. Methods Enzymol. 8, 565–571 (1966)CrossRefGoogle Scholar
  30. 30.
    Kadowaki, S., Ueda, T., Yamamoto, K., Kumagai, H., Tochikura, T.: Isolation and characterization of a blood group A substance-degrading α-N-acetylgalactosaminidase from an Acremonium sp. Agric. Biol. Chem. 53, 111–120 (1989)Google Scholar
  31. 31.
    Li, S.C., Li, Y.T.: Studies on the glycosidases of jack bean meal. 3. Crystallization and properties of beta-N-acetylhexosaminidase. J. Biol. Chem. 245, 5153–5160 (1970)PubMedGoogle Scholar
  32. 32.
    Pouria, S., Corran, P.H., Smith, A.C., Smith, H.W., Hendry, B.M., Challacombe, S.J., Tarelli, E.: Glycoform composition proWling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of Xight-mass spectrometry. Anal. Biochem. 330, 257–263 (2004)PubMedCrossRefGoogle Scholar
  33. 33.
    Matsuda, J., Kido, M., Tadano-Aritomi, K., Ishizuka, I., Tominaga, K., Toida, K., Takeda, E., Suzuki, K., Kuroda, Y.: Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse. Hum. Mol. Genet. 13, 2709–2723 (2004)PubMedCrossRefGoogle Scholar
  34. 34.
    Costello, C.E., Vath, J.E.: Tandem mass spectrometry of glycolipids. Methods Enzymol. 193, 738–768 (1990)PubMedCrossRefGoogle Scholar
  35. 35.
    Domonand, B., Costello, C.E.: A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409 (1988)CrossRefGoogle Scholar
  36. 36.
    Signorelli, P., Munoz-Olaya, J.M., Gagliostro, V., Casas, J., Ghidoni, R., Fabriàs, G.: Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells. Cancer Lett. 282, 238–243 (2009)PubMedCrossRefGoogle Scholar
  37. 37.
    Adams, J., Gross, M.L.: Charge-remote fragmentations of closed-shell Ions. A thermolytic analogy. J. Am. Chem. Soc. 111, 435–440 (1989)CrossRefGoogle Scholar
  38. 38.
    Harvey, D.J.: A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids. J. Am. Soc. Mass Spectrom. 16, 280–290 (2005)PubMedCrossRefGoogle Scholar
  39. 39.
    Qinghong, A., Adams, J.: Structure-specific collision-induced fragmentations of ceramides cationized with alkali-metal ions. Anal. Chem. 65, 7–13 (1993)CrossRefGoogle Scholar
  40. 40.
    Adams, J., Qinghong, A.: Structure determination of sphingolipids by mass spectrometry. Mass Spectrum. Rev. 12, 51–85 (1993)CrossRefGoogle Scholar
  41. 41.
    Ohashi, Y., Iwamori, M., Ogawa, T., Nagai, Y.: Analysis of long-chain bases in sphingolipids by positive ion fast atom bombardment or matrix-assisted secondary ion mass spectrometry. Biochemistry 26, 3990–3995 (1987)PubMedCrossRefGoogle Scholar
  42. 42.
    Kurogochi, M., Nishimura, S.-I.: Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097–6101 (2004)PubMedCrossRefGoogle Scholar
  43. 43.
    Stahl, B., Steup, M., Karas, M., Hillenkamp, F.: Analysis of neutral oligosaccharides by matrix-assisted laser desorption/ ionization mass spectrometry. Anal. Chem. 63, 1463–1466 (1991)CrossRefGoogle Scholar
  44. 44.
    Karlsson, K.A., Steen, G.O.: Studies on sphingosines. 13. The existence of phytosphingosine in bovine kidney sphingomyelins. Biochim. Biophys. Acta. 152, 798–800 (1968)PubMedGoogle Scholar
  45. 45.
    Breimer, M.E., Karlsson, K.A., Samuelsson, B.E.: Presence of phytosphingosine combined with 2-hydroxy fatty acids in sphingomyelins of bovine kidney and intestinal mucosa. Lipids. 10, 17–19 (1975)PubMedCrossRefGoogle Scholar
  46. 46.
    Karlsson, K.A., Nilsson, K., Samuelsson, B.E., Steen, G.O.: The presence of hydroxy fatty acids in sphingomyelins of bovine rennet stomach. Biochim. Biophys. Acta. 176, 660–663 (1969)PubMedGoogle Scholar
  47. 47.
    Kitano, Y., Iwamori, Y., Kiguchi, K., DiGiovanni, J., Takahashi, T., Kasama, K., Niwa, T., Harii, K., Iwamori, M.: Selective reduction in alpha-hydroxypalmitic acid-containing sphingomyelin and concurrent increase in hydroxylated ceramides in murine skin tumors induced by an initiation-promotion regimen. Jpn. J. Cancer Res. 87, 437–441 (1996)PubMedGoogle Scholar
  48. 48.
    Robinson, B.S., Johnson, D.W., Poulos, A.: Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa. J. Biol. Chem. 267, 1746–1751 (1992)PubMedGoogle Scholar
  49. 49.
    Morell, P., Radin, N.S.: Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid. Biochemistry 8, 506–512 (1969)PubMedCrossRefGoogle Scholar
  50. 50.
    Schaeren-Wiemers, N., van der Bijl, P., Schwab, M.E.: The UDP-galactose:ceramide galactosyltransferase: expression pattern in oligodendrocytes and Schwann cells during myelination and substrate preference for hydroxyceramide. J. Neurochem. 65, 2267–2278 (1995)PubMedCrossRefGoogle Scholar
  51. 51.
    D’Angelo, G., Polishchuk, E., Di Tullio, G., Santoro, M., Di Campli, A., Godi, A., West, G., Bielawski, J., Chuang, C.C., van der Spoel, A.C., Platt, F.M., Hannun, Y.A., Polishchuk, R., Mattjus, P., De Matteis, M.A.: Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature 449, 62–67 (2007)PubMedCrossRefGoogle Scholar
  52. 52.
    Halter, D., Neumann, S., van Dijk, S.M., Wolthoorn, J., de Mazière, A.M., Vieira, O.V., Mattjus, P., Klumperman, J., van Meer, G., Sprong, H.: Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115 (2007)PubMedCrossRefGoogle Scholar
  53. 53.
    Chatterjee, S.: Assay of lactosylceramide synthase and comments on its potential role in signal transduction. Methods Enzymol. 311, 73–81 (2000)PubMedCrossRefGoogle Scholar
  54. 54.
    Sugiura, Y., Shimma, S., Konishi, Y., Yamada, M.K., Setou, M.: Imaging mass spectrometry technology and application on ganglioside study; visualization of age-dependent accumulation of C20-ganglioside molecular species in the mouse hippocampus. PLoS One. 3, e3232 (2008)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Kouji Tanaka
    • 1
    • 2
  • Masaki Yamada
    • 3
  • Keiko Tamiya-Koizumi
    • 1
  • Reiji Kannagi
    • 1
  • Toshifumi Aoyama
    • 4
  • Atsushi Hara
    • 4
  • Mamoru Kyogashima
    • 1
    • 2
  1. 1.Division of Molecular PathologyAichi Cancer Center Research InstituteNagoyaJapan
  2. 2.Department of Oncology, Graduate School of Pharmaceutical ScienceNagoya City UniversityNagoyaJapan
  3. 3.Shimadzu CorporationKyotoJapan
  4. 4.Department of Metabolic Regulation, Institute on Aging and AdaptationShinshu University Graduate School of MedicineMatsumotoJapan

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