A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry |
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Authors: | Jin You-Xun Shi Lian Hua Yoo Hwan-Soo Lee Yong-Moon Kihara Akio Igarashi Yasuyuki So Hun-Young Yim Yong-Hyeon |
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Institution: | aCollege of Pharmacy and CBITRC, Chungbuk National University, Chongju 361-763, South Korea;bDepartment of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo 060-0812, Japan;cKorea Research Institute of Standards and Science, Daejeon 305-340, South Korea |
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Abstract: | D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI–MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for PO3]- in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay. |
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Keywords: | Sphingosine-1-phosphate Sphingosine kinase Activity F9-12 cells Electrospray ionization Tandem mass spectrometry |
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