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

研究成果: Article査読

9 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)1294-1302
ページ数9
ジャーナルJournal of Lipid Research
52
6
DOI
出版ステータスPublished - 2011 6

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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