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Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal
- Source :
- Analytical biochemistry. 408(1)
- Publication Year :
- 2010
-
Abstract
- Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography–electrospray ionization–tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25 μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC50 value of 52.4 μM.
- Subjects :
- Spectrometry, Mass, Electrospray Ionization
Electrospray ionization
Biophysics
Mass spectrometry
Biochemistry
Article
Fatty aldehyde
chemistry.chemical_compound
Mice
Biotransformation
Biosynthesis
Liquid chromatography–mass spectrometry
Sphingosine
Tandem Mass Spectrometry
Animals
Sphingosine-1-phosphate
Molecular Biology
Chromatography, High Pressure Liquid
Aldehyde-Lyases
Semicarbazones
Aldehydes
Chromatography
Fingolimod Hydrochloride
Stereoisomerism
Cell Biology
Sphingolipid
Rats
Kinetics
chemistry
Propylene Glycols
Microsomes, Liver
Hydrogenation
Lysophospholipids
Subjects
Details
- ISSN :
- 10960309
- Volume :
- 408
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Analytical biochemistry
- Accession number :
- edsair.doi.dedup.....63c1c4c709e69a041dd5f0b197c13547