Abstract
Sphingosine 1-phosphate (S1P) is a lipid metabolite and signaling molecule involved in many different physiological processes including lymphocyte circulation, T cell differentiation, antigen presentation, and maintenance of the vascular endothelial barrier. S1P is a ligand of five different G protein-coupled cell surface receptors designated S1P1-5. It has also been described as an intracellular second messenger. Quantification of S1P in biological samples is therefore an important task to decipher its signaling capabilities in vivo under physiological and pathophysiological conditions in different body fluids and organs. In this protocol, quantification of S1P is performed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS).
Keywords: Liquid chromatography, Mass spectrometry, Triple-quadrupole, Sphingolipid, Electrospray ionization
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 2. Example of S1P standard curve. For the generation of the standard curve, each concentration is measured three times. For this curve the following S1P amounts were used: 1 pmol, 3 pmol, 10 pmol, 30 pmol, 100 pmol. Figure 3. Example of S1P spectrum acquired with ESI ion source in positive mode. Representative signals of S1P and its respective internal standard C17-S1P in 10 µl of the 10 µM standard are plotted. The total amounts of S1P and C17-S1P are 100 pmol and 10 pmol, respectively. Retention times of S1P and C17-S1P are slightly different (4.93 min vs. 4.70 min).
Notes
Recipes
Acknowledgments
The extraction and measurement method is adapted from Bode and Gräler (2012).
References
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