title: A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples.
abstract: PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.
authors: Inserm, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
authors: Gaëtan
authors: G
authors: Chicanne
authors: Sonia
authors: S
authors: Severin
authors: Cécile
authors: C
authors: Boscheron
authors: Anne-Dominique
authors: AD
authors: Terrisse
authors: Marie-Pierre
authors: MP
authors: Gratacap
authors: Frédérique
authors: F
authors: Gaits-Iacovoni
authors: Hélène
authors: H
authors: Tronchère
authors: Bernard
authors: B
authors: Payrastre
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D058546:Class III Phosphatidylinositol 3-Kinases; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C055525:phosphatidylinositol 3-phosphate; C492139:PIKFYVE protein, human