title: Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ).
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.
authors.affiliation: Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Takuji
authors.initials: T
authors.lastname: Nabetani
authors.affiliation: Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Makino
authors.affiliation: Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan; INSERM U1060, université Lyon1, INSA-Lyon, 69621 Villeurbanne, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Hullin-Matsuda
authors.affiliation: Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan; Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Taka-Aki
authors.initials: TA
authors.lastname: Hirakawa
authors.affiliation: Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University (TWIns), Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takeoka
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
authors.affiliation: Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan; INSERM U1060, université Lyon1, INSA-Lyon, 69621 Villeurbanne, France. Electronic address: kobayasi@riken.jp.
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Laboratory for Molecular Membrane Neuroscience, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hirabayashi
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000588:Amines / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004285:Dogs; D057166:High-Throughput Screening Assays; D006801:Humans; D006868:Hydrolysis; D015398:Signal Transduction; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D000588:Amines; D002518:Ceramides; D003432:Cross-Linking Reagents; D003902:Detergents; D013388:Succinimides; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase; C001426:N-hydroxysuccinimide