Determination of sphingosine kinase activity in biological samples by liquid chromatography-tandem mass spectrometry.
Sphingosine kinase (SphK) is a key enzyme in modulating the levels of sphingosine 1-phosphate (S1P) as well as an important enzyme in numerous biological responses. Using C17-sphingosine as a substrate, we established a rapid, sensitive and highly efficient method for determination of SphK activity by analyzing the product C17-sphingosine 1-phosphate (C17-S1P) using liquid chromatography-tandem mass spectrometry. The standard curve for C17-S1P was linear over a wide range (10-1000 ng/mL) with correlation coefficient (r(2)) greater than 0.999. The lower limit of quantification for C17-S1P was 10 ng/mL. The K(m) values for C17-sphingosine and ATP were determined to be 28.17 and 188.5  microM, respectively. More importantly, the SphK activity dramatically increased in cultured HEK 293 cells expressing wild-type SphK1 as well as cells treated with tumor necrosis factor-alpha, a sphingosine kinase activator. In contrast, the SphK activity decreased in cultured HEK 293 cells treated with dimethylsphngosine, a sphingosine kinase inhibitor. In conclusion, this method was sensitive and rapid in the determination of SphK acitivity, providing striking utilities in exploring the sphingosine kinase signaling pathway and screening active compounds targeting SphK activity.
Lan
Tian
T
Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Bi
Huichang
H
Xu
Suowen
S
Le
Kang
K
Xie
Zhiyong
Z
Liu
Yiwei
Y
Huang
Heqing
H
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000704:Analysis of Variance; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D017711:Nonlinear Dynamics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000097:blood / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099157:Sphingosine Kinase
D000255:Adenosine Triphosphate; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D014409:Tumor Necrosis Factor-alpha; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C061800:N,N-dimethylsphingosine