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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)


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
Issue number6
Publication statusPublished - 2011 Jun


  • 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
  • Endocrinology
  • Cell Biology


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