Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)

Takuji Nabetani, Asami Makino, Françoise Hullin-Matsuda, Taka Aki Hirakawa, Shinji Takeoka, Nozomu Okino, Makoto Ito, Toshihide Kobayashi, Yoshio Hirabayashi

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    Abstract

    Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl) pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.

    Original languageEnglish
    Pages (from-to)1294-1302
    Number of pages9
    JournalJournal of Lipid Research
    Volume52
    Issue number6
    DOIs
    Publication statusPublished - 2011 Jun

    Fingerprint

    Sphingolipids
    Ceramides
    Amines
    Sphingomyelin Phosphodiesterase
    Propane
    Madin Darby Canine Kidney Cells
    Jurkat Cells
    Detergents
    Labeling
    Mass spectrometry
    Hydrolysis
    Mass Spectrometry
    Apoptosis
    Lipids
    Proteins
    ceramide monohexoside

    Keywords

    • Apoptosis
    • Ceramide
    • Cleavable detergent
    • Isobaric tags for relative and absolute quantitation
    • Mass spectrometry
    • Monohexosylceramide
    • Multiple reaction monitoring
    • Sphingolipid ceramide N-deacylase
    • Sphingomyelinase

    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology
    • Endocrinology

    Cite this

    Nabetani, T., Makino, A., Hullin-Matsuda, F., Hirakawa, T. A., Takeoka, S., Okino, N., ... Hirabayashi, Y. (2011). Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). Journal of Lipid Research, 52(6), 1294-1302. https://doi.org/10.1194/jlr.D014621

    Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). / Nabetani, Takuji; Makino, Asami; Hullin-Matsuda, Françoise; Hirakawa, Taka Aki; Takeoka, Shinji; Okino, Nozomu; Ito, Makoto; Kobayashi, Toshihide; Hirabayashi, Yoshio.

    In: Journal of Lipid Research, Vol. 52, No. 6, 06.2011, p. 1294-1302.

    Research output: Contribution to journalArticle

    Nabetani, T, Makino, A, Hullin-Matsuda, F, Hirakawa, TA, Takeoka, S, Okino, N, Ito, M, Kobayashi, T & Hirabayashi, Y 2011, 'Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ)', Journal of Lipid Research, vol. 52, no. 6, pp. 1294-1302. https://doi.org/10.1194/jlr.D014621
    Nabetani T, Makino A, Hullin-Matsuda F, Hirakawa TA, Takeoka S, Okino N et al. Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). Journal of Lipid Research. 2011 Jun;52(6):1294-1302. https://doi.org/10.1194/jlr.D014621
    Nabetani, Takuji ; Makino, Asami ; Hullin-Matsuda, Françoise ; Hirakawa, Taka Aki ; Takeoka, Shinji ; Okino, Nozomu ; Ito, Makoto ; Kobayashi, Toshihide ; Hirabayashi, Yoshio. / Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ). In: Journal of Lipid Research. 2011 ; Vol. 52, No. 6. pp. 1294-1302.
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