Analysis of intact phosphoinositides in biological samples

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P...

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Vydané v:Journal of lipid research Ročník 47; číslo 7; s. 1588 - 1596
Hlavní autori: Pettitt, Trevor R, Dove, Stephen K, Lubben, Anneke, Calaminus, Simon D J, Wakelam, Michael J O
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States Elsevier 01.07.2006
Predmet:
Blood Platelets - chemistry
Chromatography, Liquid - methods
Chromatography, Liquid - statistics & numerical data
Humans
lipidomics
liquid chromatography-mass spectrometry
mass spectrometry
Mass Spectrometry - methods
Mass Spectrometry - statistics & numerical data
Phosphatidylinositols - analysis
Phosphatidylinositols - blood
Phosphatidylinositols - chemistry
platelets
Saccharomyces cerevisiae - chemistry
Sensitivity and Specificity
yeast
ISSN:0022-2275
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Popis
Shrnutí:It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.
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ISSN:0022-2275
DOI:10.1194/jlr.d600004-jlr200