A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry

Jin, You-Xun; Shi, Lian Hua; Yoo, Hwan‐Soo; Lee, Young Moo; Kihara, Akio; Igarashi, Yasuyuki; So, Hun-Young; Yim, Yong‐Hyeon

doi:10.1016/j.ab.2008.05.021

Cited by 8 publications

(9 citation statements)

References 23 publications

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“…Fig. 5 demonstrates that intracellular S1P content increased in response to TNF-␣ treatment; whereas intracellular S1P content decreased in response to DMS treatment, which is consistent with previous data [25][26][27][28][29]. Table 4 shows the quantified levels of Sph and S1P in HEK 293 cells, mouse kidney and human plasma.…”

Section: Application Of the Methods In Biological Samplessupporting

confidence: 93%

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

Lan¹,

Bi²,

Liu³

et al. 2011

Journal of Chromatography B

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Section: Application Of the Methods In Biological Samplessupporting

confidence: 93%

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

Lan¹,

Bi²,

Liu³

et al. 2011

Journal of Chromatography B

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“…To validate the sensitivity of this assay and further develop a method of screening agents targeting SphK, we measured SphK activity in extracts of HEK 293 cells treated with TNFa for 10 min or DMS for 1 h. Concurrent with previous data (Jin et al , 2008; Xia et al , 1998), TNFa stimulated endogenous SphK activity up to 2.5‐fold in cells, whereas DMS treatment resulted in a remarkable decrease in SphK activity (Fig. 7).…”

Section: Resultssupporting

confidence: 60%

“…Several analytical methods have been reported for the measurement of SphK activity in biological samples such as radioactive assays (Olivera et al , 1998, 2000; Vessey et al , 2005), high‐performance liquid chromatography (HPLC) (Billich and Ettmayer, 2004; Caligan et al , 2000; Min et al , 2002) and mass spectrometry (Jin et al , 2008). Conventionally, most of the assays rely on the use of radiolabeled substrate ([g‐ 32 P]ATP or [ 3 H]sphingoid base), and the use of organic solvents to extract the [g‐ 32 P]S1P or [ 3 H]S1P, and the analytes are subsequently separated by thin‐layer chromatography (TLC).…”

Section: Introductionmentioning

confidence: 99%

“…The newly available technology of liquid chromatography–tandem mass spectroscopy (LC‐MS/MS) proves to be one of the most sensitive methods for determining many different sphingolipids simultaneously, and S1P is no exception (Berdyshev et al , 2005; Bielawski et al , 2006). More recently, SphK activity assays using direct infusion electrospray ionization tandem mass spectrometry have been developed (Jin et al , 2008). This method has obvious advantages, including higher efficiency and selectivity as well as simultaneous quantification capability of various sphingolipids.…”

Section: Introductionmentioning

confidence: 99%

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Determination of sphingosine kinase activity in biological samples by liquid chromatography–tandem mass spectrometry

Lan

et al. 2010

Biomedical Chromatography

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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.

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“…This led us to examine whether these color changes were quantifi able and SphK dependent. In the presence of SphK1 but ( 13,14 ). For many researchers, this is not a cost-effective alternative for high-throughput screening of small-molecule libraries as it also requires highly specialized expertise and equipment.…”

Section: Sphk-dependent Changes In Visible Spectra Of Nbd-sphmentioning

confidence: 99%

A real-time high-throughput fluorescence assay for sphingosine kinases

Lima

Milstien

Spiegel

2014

Journal of Lipid Research

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Currently, there are a wide variety of assays that are useful for the characterization of SphK activity in cell extracts or of the recombinant enzymes. However, most lack the capacity to follow SphK activity in real time, precluding the convenient characterization of SphK mutants, competitors, or inhibitors in a high-throughput or "one-pot" approach. The most widely cited method was originally developed by our group and utilizes [ ␥ -32 P]ATP and D-erythroSph as substrates, and it requires differential organic extraction, TLC separation, and quantifi cation of the radiolabeled S1P product ( 4 ). This method is highly accurate but is labor intensive and not high throughput. More specialized variations of the radiolabeling assay have been developed. For example, those that utilize [ 3 H]Sph as a substrate ( 5 ) or biotinylated-Sph with [ ␥ -32 P]ATP and capture of the radiolabeled phosphorylated product with streptavidin ( 6 ), or a method that conveniently avoids organic extraction and TLC separation and quantifi es [ 32 P] S1P by scintillation proximity counting ( 7 ). However, as with the classic [ ␥ -32 P]ATP method, these are not useful for monitoring reactions in real time and also have the drawback of requiring radioactive materials.Fluorophore-labeled Sphs have also been extensively used as substrates for examining SphK activity ( 8, 9 ) and have been shown to be useful surrogates for Sph in the screening of inhibitors. However, these are not highthroughput assays because in order to measure enzymatic activity the phosphorylated products must be isolated by organic fractionation ( 10 ) or processed with specialized equipment by capillary electrophoresis ( 9 ) or HPLC ( 11 ).A bioluminescence assay has been developed to measure ADP produced when the ␥ -phosphate from ATP is transferred to Sph ( 12 ). However, this is an indirect measure of SphK activity through ATP hydrolysis rates. Among the most accurate methods are those that fractionate and Sphingosine kinases (SphK), consisting of two isoforms, SphK1 and SphK2, catalyze the formation of sphingosine-1-phosphate (S1P) by transferring the ␥ -phosphate from ATP to the primary hydroxyl of sphingosine (Sph). S1P is a potent bioactive lipid that can regulate many complex cellular processes, including growth, survival, migration, angiogenesis, and infl ammation, to name a few ( 1-3 ). Accordingly, abnormal regulation of SphKs that leads to elevated levels of S1P has been implicated in the etiology of cancer, as well as autoimmune and cardiovascular diseases ( 3 ). Hence, pharmacologically modulating SphK activity has important therapeutic potential, and technologies that can assist in the discovery of SphK inhibitors are critical tools for their development.

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A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry

Cited by 8 publications

References 23 publications

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

Determination of sphingosine kinase activity in biological samples by liquid chromatography–tandem mass spectrometry

A real-time high-throughput fluorescence assay for sphingosine kinases

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