A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry.
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 [PO(3)](-) 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.
College of Pharmacy and CBITRC, Chungbuk National University, Chongju 361-763, South Korea.
You-Xun
YX
Jin
Lian Hua
LH
Shi
Hwan-Soo
HS
Yoo
Yong-Moon
YM
Lee
Akio
A
Kihara
Yasuyuki
Y
Igarashi
Hun-Young
HY
So
Yong-Hyeon
YH
Yim
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D000099157:Sphingosine Kinase
D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D000099157:Sphingosine Kinase; C016658:phorbolol myristate acetate; C061800:N,N-dimethylsphingosine