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Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species
Published on: May 7, 2018
Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ).
Takuji Nabetani1, Asami Makino1, Françoise Hullin-Matsuda2
1Lipid Biology Laboratory, Advanced Science Institute, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan.
Journal of Lipid Research
|April 14, 2011
Summary
This study introduces a novel method using isobaric tags for relative and absolute quantitation (iTRAQ) to simultaneously measure ceramides and monohexosylceramides. This technique enhances the sensitive detection of these vital signaling lipids in biological samples.
Area of Science:
- Lipidomics
- Biochemistry
- Cell Signaling
Background:
- Ceramides are critical signaling lipids involved in cell differentiation, senescence, proliferation, and apoptosis.
- Accurate quantification of ceramides is challenging due to their low abundance.
- Existing methods like iTRAQ are effective for proteins and aminophospholipids but not yet for sphingolipids.
Purpose of the Study:
- To develop and validate a novel method for simultaneous measurement of multiple ceramide and monohexosylceramide samples.
- To adapt the iTRAQ labeling technique for the accurate quantification of sphingolipids.
- To apply the developed method for profiling ceramides in cellular models.
Main Methods:
- Hydrolysis of sphingolipids using sphingolipid ceramide N-deacylase (SCDase) to expose free amino groups.
- Conjugation of iTRAQ reagent to the exposed amino groups of sphingolipids.
- Use of a cleavable detergent (PPS) to improve hydrolysis and mass spectrometry accuracy.
Main Results:
- Successful simultaneous measurement of multiple ceramide and monohexosylceramide samples.
- Demonstrated application of iTRAQ labeling for sphingolipid determination.
- Accurate profiling of ceramides and monohexosylceramides in sphingomyelinase-treated MDCK cells and apoptotic Jurkat cells.
Conclusions:
- The developed iTRAQ-based method enables sensitive and simultaneous quantification of ceramides and monohexosylceramides.
- This approach overcomes previous limitations in sphingolipid analysis.
- The method is applicable for comprehensive ceramide profiling in various biological contexts, including disease models.
