Separation of fluorescent phosphatidyl inositol phosphates by CE

Mwongela, Simon M.; Lee, Katherine; Sims, Christopher E.; Allbritton, Nancy L.

doi:10.1002/elps.200600594

Cited by 16 publications

(25 citation statements)

References 40 publications

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“…CE analysis of lipid analytes was performed using a custom-built CE system with laser-induced fluorescence detection as previously described 21 . Fused-silica capillaries (40 cm length, 50 μm i.d., 360 μm o.d., Polymicro Technologies, Phoenix, AZ) were used for the analyte separations.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, Caliper Lifesciences employed FL-PIP 2 as a PI3K substrate for an in vitro assay in which conversion of the FL-PIP 2 to FL-PIP 3 was monitored by electrophoretic chemical separation with laser-induced fluorescence detection in a microfabricated fluidic chip. Such highly-sensitive, chemical separation techniques for monitoring phosphorylation lend themselves to cell-based assays, and several examples using capillary electrophoresis (CE) with fluorescent peptide- and lipid-based probes have been reported in single-cell biochemical measurements 20–21 . However, the amphiphilic nature of PI lipids renders them to potential loss on the column during CE separation.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives

et al. 2011

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PI3K signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP2 derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives

et al. 2011

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“…46 The efficiency and resolution of the separation of SphFl and SphFl-1-P were similar to those of other CE separations of lipids. [47][48][49] …”

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confidence: 99%

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

et al. 2008

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Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...

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“…CE with indirect UV detection was used to determine lysophosphatidic acids, anionic phospholipids and phospholipid classes [13][14][15][16][17]. CE with fluorescence detection was used to study cardiolipin, phosphatidylserine and sphingolipids [18][19][20][21][22][23]. CE in conjunction with MS was used to analyze phosphatidylethanol, lyso-glycerophosphocholine and other phospholipids [9,12,24].…”

Section: Introductionmentioning

confidence: 99%

Micellar electrokinetic chromatography profiles of human high‐density lipoprotein phospholipids

et al. 2011

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A simple and fast micellar electrokinetic chromatography (MEKC) method was developed to investigate phospholipids isolated from human high-density lipoproteins (HDL). To optimize the MEKC conditions, several factors including bile salt concentration and organic modifier concentration in the separation buffer as well as temperature have been examined. The optimal separation buffer chosen was a mixture of 50 mM bile salts, 30% v/v 1-propanol and 10 mM sodium phosphate (pH 8.5). The applied voltage and temperature selected were 25 kV and 40°C, respectively. Meanwhile, high-salt stacking has been performed for sample pre-concentration to enhance peak sensitivity. Several factors including organic modifier concentration and salt concentration in the sample matrix as well as sample injection time have been optimized. The optimal sample buffer selected was a mixture of 100 mM NaCl and 20% 1-propanol, and the optimal sample injection time selected was 32 s under a pressure of 0.5 psi. Several phospholipid standards including lysophosphatidyl choline, phosphatidyl choline (PC), sphingomyelin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and phosphatidic acid have been studied using the optimal MEKC method. The MEKC profile of the mixed phospholipid standards showed good separation and reproducibility. The linear ranges for PC and sphingomyelin were 0.025-1.2 and 0.025-2.0 mg/mL, respectively. The concentration limits of detection of PC and sphingomyelin were 0.0156 and 0.0199 mg/mL, respectively. Using phosphatidic acid as an internal standard, precision and accuracy have been measured for PC and sphingomyelin. The intraday and interday quantitative analysis showed good results. The new MEKC method has been used to characterize native, in vitro oxidized and glycated human HDL phospholipids within 16 min. At absorbance 200 nm, two similar peaks were observed for native and oxidized HDL phospholipids, but three peaks were observed for glycated HDL phospholipids. Interestingly, at absorbance 234 nm, distinctively different MEKC profiles were observed for the three HDL phospholipids.

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Separation of fluorescent phosphatidyl inositol phosphates by CE

Cited by 16 publications

References 40 publications

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

Micellar electrokinetic chromatography profiles of human high‐density lipoprotein phospholipids

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