Quantitative structural characterization of phosphatidylinositol phosphates from biological samples

Abstract

The aspects of cellular metabolism controlled by phosphatidylinositol phosphates (PtdInsPs) have been broadly expanded, and these phospholipids have drawn tremendous attention as pleiotropic signaling molecules. PtdInsPs analysis using LC/MS/MS has remained challenging due to the strong hydrophilicity of these lipids. Multiple reaction monitoring (MRM) or a neutral loss scan has been performed to quantitatively measure PtdInsPs after chemical derivatization on the phosphate groups of inositol moieties. Only predefined PtdInsPs can be measured in MRM mode, and fatty acyl compositions of sn-1 and sn-2 positions of PtdInsPs cannot be obtained from a neutral loss scan. In our present study, we developed a simple LC/MS/MS method for structural identification of sn-1 and sn-2 fatty acids of PtdInsPs and their relative quantitation. Precursor ion scans of sn-1 monoacylglycerols (MAGs) of PtdInsPs provided structural information about the lipids, and ammonium adduction enhanced signal intensities of PtdInsPs. The relative amount of observed PtdInsPs in biological samples could be compared using chromatographic peak areas from the neutral loss scans. Using precursor ion scans of sn-1 MAG and neutral loss scans of headgroups, major PtdInsPs in cells and tissues were successfully identified with structural information of sn-1 and sn-2 fatty acids, and their relative amounts in different samples were compared. The aspects of cellular metabolism controlled by phosphatidylinositol phosphates (PtdInsPs) have been broadly expanded, and these phospholipids have drawn tremendous attention as pleiotropic signaling molecules. PtdInsPs analysis using LC/MS/MS has remained challenging due to the strong hydrophilicity of these lipids. Multiple reaction monitoring (MRM) or a neutral loss scan has been performed to quantitatively measure PtdInsPs after chemical derivatization on the phosphate groups of inositol moieties. Only predefined PtdInsPs can be measured in MRM mode, and fatty acyl compositions of sn-1 and sn-2 positions of PtdInsPs cannot be obtained from a neutral loss scan. In our present study, we developed a simple LC/MS/MS method for structural identification of sn-1 and sn-2 fatty acids of PtdInsPs and their relative quantitation. Precursor ion scans of sn-1 monoacylglycerols (MAGs) of PtdInsPs provided structural information about the lipids, and ammonium adduction enhanced signal intensities of PtdInsPs. The relative amount of observed PtdInsPs in biological samples could be compared using chromatographic peak areas from the neutral loss scans. Using precursor ion scans of sn-1 MAG and neutral loss scans of headgroups, major PtdInsPs in cells and tissues were successfully identified with structural information of sn-1 and sn-2 fatty acids, and their relative amounts in different samples were compared. Phosphatidylinositol phosphates (PtdInsPs), a type of cellular phospholipids, play an important role in signal transduction, membrane dynamics, and cell proliferation (1.Toker A. Phosphoinositides and signal transduction.Cell. Mol. Life Sci. 2002; 59: 761-779Crossref PubMed Scopus (178) Google Scholar, 2.Mayinger P. Phosphoinositides and vesicular membrane traffic.Biochim. Biophys. Acta. 2012; 1821: 1104-1113Crossref PubMed Scopus (84) Google Scholar, 3.Okada M. Jang S.W. Ye K. Akt phosphorylation and nuclear phosphoinositide association mediate mRNA export and cell proliferation activities by ALY.Proc. Natl. Acad. Sci. USA. 2008; 105: 8649-8654Crossref PubMed Scopus (69) Google Scholar, 4.Rajala R.V. Rajala A. Morris A.J. Anderson R.E. Phosphoinositides: minor lipids make a major impact on photoreceptor cell functions.Sci. Rep. 2014; 4: 5463Crossref PubMed Scopus (7) Google Scholar). The aspects of cellular metabolism controlled by PtdInsPs have been broadly expanded, and PtdInsPs have drawn tremendous attention as pleiotropic signaling molecules for eukaryotic cells in a wide range of biomedical research (5.Vivanco I. Sawyers C.L. The phosphatidylinositol 3-kinase AKT pathway in human cancer.Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5130) Google Scholar, 6.Kim I. Kim H.G. So J.N. Kim J.H. Kwak H.J. Koh G.Y. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway.Circ. Res. 2000; 86: 24-29Crossref PubMed Scopus (519) Google Scholar). This class of phospholipids regulates biology by controlling vesicular trafficking and also by modulating lipid distribution and metabolism via close interaction with proteins (7.Di Paolo G. De Camilli P. Phosphoinositides in cell regulation and membrane dynamics.Nature. 2006; 443: 651-657Crossref PubMed Scopus (2069) Google Scholar, 8.Krauss M. Haucke V. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling.EMBO Rep. 2007; 8: 241-246Crossref PubMed Scopus (105) Google Scholar, 9.Engelman J.A. Luo J. Cantley L.C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism.Nat. Rev. Genet. 2006; 7: 606-619Crossref PubMed Scopus (2508) Google Scholar). Dysregulation of PtdInsPs is known to be involved in various human diseases, such as cancer, obesity, and diabetes (10.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (970) Google Scholar, 11.Manna P. Jain S.K. Phosphatidylinositol-3,4,5-triphosphate and cellular signaling: implications for obesity and diabetes.Cell. Physiol. Biochem. 2015; 35: 1253-1275Crossref PubMed Scopus (48) Google Scholar). For example, the relationship between phosphatidylinositol 3-kinase (PI3K) and oncogenic receptor tyrosine kinases puts an emphasis on the role of PtdInsPs in cancer biology. PtdInsPs promote cell proliferation and prevent apoptosis; thus, they are known as representative oncogenic lipids (10.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (970) Google Scholar). PtdInsPs are generated metabolically via phosphorylation on the hydroxyl groups of the inositol sugar of phosphatidylinositol (PtdIns). This phosphorylation is regulated by the enzymatic action of several kinases and phosphatases. In general, monophosphorylation of PtdIns occurs in endomembranes such as the Golgi, and di- or triphosphorylation occurs mainly in plasma membranes (10.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (970) Google Scholar). PtdInsPs exist in various cells and tissues, and their amount shows significant variation (12.Nasuhoglu C. Feng S. Mao J. Yamamoto M. Yin H.L. Earnest S. Barylko B. Albanesi J.P. Hilgemann D.W. Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection.Anal. Biochem. 2002; 301: 243-254Crossref PubMed Scopus (112) Google Scholar, 13.Wenk M.R. Lucast L. Di Paolo G. Romanelli A.J. Suchy S.F. Nussbaum R.L. Cline G.W. Shulman G.I. McMurray W. De Camilli P. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.Nat. Biotechnol. 2003; 21: 813-817Crossref PubMed Scopus (204) Google Scholar). PtdIns and PtdInsPs are low-abundance lipid classes consisting of 10%–20% of total cellular phospholipids. Phosphatidylinositol bis- (PtdInsP2) or triphosphates (PtdInsP3) constitute less than 1% of the total of PtdIns and PtdInsPs (14.Kielkowska A. Niewczas I. Anderson K.E. Durrant T.N. Clark J. Stephens L.R. Hawkins P.T. A new approach to measuring phosphoinositides in cells by mass spectrometry.Adv. Biol. Regul. 2014; 54: 131-141Crossref PubMed Scopus (58) Google Scholar, 15.Stephens L.R. Jackson T.R. Hawkins P.T. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system?.Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). The amount of PtdInsP3 contributes 2%–5% of PtdInsP2 or less than 0.25% of the inositol lipids in eukaryotic cell membranes (10.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (970) Google Scholar, 16.Milne S.B. Ivanova P.T. DeCamp D. Hsueh R.C. Brown H.A. A targeted mass spectrometric analysis of phosphatidylinositol phosphate species.J. Lipid Res. 2005; 46: 1796-1802Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Observing PtdInsP3 in biological samples is difficult, even upon stimulation (17.Haag M. Schmidt A. Sachsenheimer T. Brugger B. Quantification of signaling lipids by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS).Metabolites. 2012; 2: 57-76Crossref PubMed Scopus (36) Google Scholar). PtdInsPs have been deacylated and analyzed by measuring 32P-labeled inositides or 3H-myo-inositol using HPLC (18.Alter C.A. Wolf B.A. Identification of phosphatidylinositol 3,4,5-trisphosphate in pancreatic islets and insulin-secreting beta-cells.Biochem. Biophys. Res. Commun. 1995; 208: 190-197Crossref PubMed Scopus (33) Google Scholar, 19.Ho C.Y. Choy C.H. Botelho R.J. Radiolabeling and quantification of cellular levels of phosphoinositides by high performance liquid chromatography-coupled flow scintillation.J. Vis. Exp. 2016; 107: e53529Google Scholar, 20.Hama H. Torabinejad J. Prestwich G.D. DeWald D.B. Measurement and immunofluorescence of cellular phosphoinositides.Methods Mol. Biol. 2004; 284: 243-258PubMed Google Scholar). TLC has been conventionally used for the analysis of phospholipids and has been applied to analyze isomers of PtdInsPs (21.Hegewald H. One-dimensional thin-layer chromatography of all known D-3 and D-4 isomers of phosphoinositides.Anal. Biochem. 1996; 242: 152-155Crossref PubMed Scopus (23) Google Scholar, 22.Furutani M. Itoh T. Ijuin T. Tsujita K. Takenawa T. Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides.J. Biochem. 2006; 139: 663-670Crossref PubMed Scopus (14) Google Scholar). A TLC analysis is relatively nonspecific, and lipids extracted from a TLC plate have been further analyzed with GC or GC/MS (23.König S. Hoffmann M. Mosblech A. Heilmann I. Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography.Anal. Biochem. 2008; 378: 197-201Crossref PubMed Scopus (44) Google Scholar). These methods using radiolabeling with 32P or a TLC separation are very laborious and have limited applications. Mass spectrometric detection of phospholipids has been performed in a positive or negative ion mode. Collision-induced dissociation (CID) is the most commonly used tandem mass spectrometric strategy. CID in a positive ion mode provides structural information about the polar headgroups of phospholipids. Haag (17.Haag M. Schmidt A. Sachsenheimer T. Brugger B. Quantification of signaling lipids by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS).Metabolites. 2012; 2: 57-76Crossref PubMed Scopus (36) Google PtdInsPs from human cells and after stimulation in a positive ion mode, through from neutral loss of the from PtdInsPs. the other the fatty acid composition at the sn-1 or sn-2 of the phospholipids obtained from negative ion mode research groups have performed structural of PtdInsPs using CID in negative ion mode, and fatty acyl compositions using the neutral loss of fatty acid or M.R. Lucast L. Di Paolo G. Romanelli A.J. Suchy S.F. Nussbaum R.L. Cline G.W. Shulman G.I. McMurray W. De Camilli P. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.Nat. Biotechnol. 2003; 21: 813-817Crossref PubMed Scopus (204) Google Scholar, 16.Milne S.B. Ivanova P.T. DeCamp D. Hsueh R.C. Brown H.A. A targeted mass spectrometric analysis of phosphatidylinositol phosphate species.J. 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PubMed Scopus Google Scholar, in an and Biophys. Acta. 2002; PubMed Scopus Google Scholar, H. method for in species levels growth in 2008; PubMed Scopus Google Scholar). LC/MS/MS is to phospholipids or chemical derivatization and provides structural information for the identification of phospholipids. groups on the inositol chemical on PtdInsPs compared with other of phospholipids. Observing PtdInsPs a mass spectrometric analysis is due to the of structural using a tandem mass spectrometric analysis for PtdInsPs have been using the M.R. Lucast L. Di Paolo G. Romanelli A.J. Suchy S.F. Nussbaum R.L. Cline G.W. Shulman G.I. McMurray W. De Camilli P. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.Nat. Biotechnol. 2003; 21: 813-817Crossref PubMed Scopus (204) Google Scholar, 16.Milne S.B. Ivanova P.T. DeCamp D. Hsueh R.C. Brown H.A. A targeted mass spectrometric analysis of phosphatidylinositol phosphate species.J. Lipid Res. 2005; 46: 1796-1802Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, J. of and by electrospray ionization tandem mass a Mass 2000; PubMed Scopus Google Scholar, H. method for in species levels growth in 2008; PubMed Scopus Google Scholar). the electrospray ionization could be for PtdInsPs they are lipids in biological with an separation could be a method for analysis of PtdInsPs from various biological samples to isomers and to ion liquid chromatography separation has in performance due to the high of the phosphate groups on PtdInsPs. H. method for in species levels growth in 2008; PubMed Scopus Google separation of PtdInsPs using strong In a has been to polar PtdInsPs T.R. Phosphoinositide analysis by liquid Mol. Biol. PubMed Scopus Google Scholar, T.R. S.K. A. of phosphoinositides in biological Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). has been used to acids or phosphates (14.Kielkowska A. Niewczas I. Anderson K.E. Durrant T.N. Clark J. Stephens L.R. Hawkins P.T. A new approach to measuring phosphoinositides in cells by mass spectrometry.Adv. Biol. Regul. 2014; 54: 131-141Crossref PubMed Scopus (58) Google Scholar, T. of of acids by 2007; 46: PubMed Scopus Google Scholar, H. using of groups and of phosphate groups via reaction with in mass spectrometry via a positive 2014; 86: Google Scholar). H. using of groups and of phosphate groups via reaction with in mass spectrometry via a positive 2014; 86: Google enhanced the ionization and of and provided than lipids, signal due to ionization after In of has in chromatographic peak and compared with of species S. M. T. profiling of polar lipids by mass spectrometry with A. 2013; PubMed Scopus Google Scholar). of the phosphate groups of the inositol could an separation and ionization of as as prevent of PtdInsPs of phosphate groups be for the analysis of PtdInsP2 and PtdInsP3 J. Anderson K.E. V. Stephens L.R. Hawkins P.T. Quantification of PtdInsP3 species in cells and tissues by mass spectrometry.Nat. 8: PubMed Scopus Google exist in very levels in biological PtdInsPs from cells have been measured using a neutral loss scan upon in the positive ion mode after derivatization with (14.Kielkowska A. Niewczas I. Anderson K.E. Durrant T.N. Clark J. Stephens L.R. Hawkins P.T. A new approach to measuring phosphoinositides in cells by mass spectrometry.Adv. Biol. Regul. 2014; 54: 131-141Crossref PubMed Scopus (58) Google Scholar). Clark J. Anderson K.E. V. Stephens L.R. Hawkins P.T. Quantification of PtdInsP3 species in cells and tissues by mass spectrometry.Nat. 8: PubMed Scopus Google successfully PtdInsP3 with other PtdInsPs from cells and tissues using LC/MS/MS after performed analysis for PtdInsPs to the information of the sn-1 and sn-2 fatty acyl compositions of the observed PtdInsPs. of PtdInsPs successfully detection of PtdInsPs in biological using LC/MS/MS at the of detection for of have detection can be by using ammonium This be to PtdInsPs using neutral loss scans to for PtdInsPs in biological the in scan mode is than in such as reaction In be for precursor ion scans of sn-1 in positive ion mode to fatty acid compositions at sn-1 and sn-2 positions of PtdInsPs. In study, the of precursor ion scans of sn-1 MAG and neutral loss scans of headgroups successfully applied to the relative amounts of PtdInsPs in biological and were used to the of PtdInsPs after PtdInsPs were from The PtdInsPs were the PtdIns PtdIns and were in and at and were from or PtdInsPs from biological samples were extracted by using the method with minor A method of total lipid and J. Biochem. Physiol. PubMed Scopus Google Scholar). of and of of PtdInsPs in were to a cells or of were using a and to for at to all in the for the of PtdInsPs. of and of were to the by The layer the lipids and to a new of to the and the layer and to the The layer PtdInsPs The at to chemical of PtdInsPs were using the as for biological The in and of in The derivatization reaction to for at and acid to the reaction to the reaction and the the layer of the and The in for the LC/MS/MS The cell cells were from the were in of with and were in For the cells were in with or or for the the cells were and with The cells were with and at were at with and in the at the of with to and and were to the and by the of and at of were with or via the after the and in liquid and at LC/MS/MS with HPLC and a in the positive ion mode with of using and gas at a of The separation for PtdInsPs used A ammonium and ammonium and at and The separation as of at of for to of for of for and to of for For A and were also used with the A neutral loss scan and a precursor ion to the structural of were performed in the positive ion mode. of were used for the neutral loss scans on neutral loss and used for the precursor ion scans. The extracted ion to the lipid used for relative after using the LC/MS/MS were analyzed with of the and PtdInsP3 were used to the PtdInsPs different of PtdInsPs were via chemical derivatization with with at for PtdInsPs. the phosphate hydroxyl groups were known to be J. Anderson K.E. V. Stephens L.R. Hawkins P.T. Quantification of PtdInsP3 species in cells and tissues by mass spectrometry.Nat. 8: PubMed Scopus Google Scholar). phosphate groups were after the reaction in our For example, PtdInsP2 hydroxyl groups on a phosphate to the PtdInsP2 mainly PtdInsPs were as at such as or is commonly observed in mass spectrometric ionization of these be and the analysis and of observed or ion for PtdInsPs. the signal intensities of ammonium with PtdInsPs were to of in the positive ion mode. shows the mass of PtdInsPs ammonium ion in the the most ion for PtdInsPs as in the most ion for PtdInsPs species than the ammonium ion in the and ammonium ion enhanced signal intensities for PtdInsPs. ammonium ion the for signal and ammonium ion adduction the we compared of and The major ion in CID of or in to the most to MAG also observed and from further at the of the sn-2 This provided structural information fatty acids at the sn-1 of PtdInsPs. the between and the for other PtdInsPs ammonium of PtdInsPs were used for structural of PtdInsPs in The neutral loss scans of headgroups could and PtdInsPs from as in PtdInsPs were at different from and PtdInsPs. The used as were also at The of of inositol phosphates the PtdInsPs with the fatty acyl PtdInsPs species were observed ammonium ion in the the PtdInsPs were the signal due to ammonium ion adduction between and on PtdInsPs The of of ammonium ion in for signal and analysis by several groups using method for the of in using a between MRM and 2012; PubMed Scopus Google Scholar, G. of of ionization on Mass 2004; PubMed Scopus Google Scholar). to PtdInsPs from biological samples such as cells and and also to the relative of PtdInsPs. and signal of ammonium ion PtdInsPs were from due to ammonium ion adduction using peak areas of ammonium PtdInsPs to PtdInsPs in to signal due to ammonium ion ammonium ion of PtdInsPs and the relative for type of PtdInsPs in of neutral loss scans of as in ion of PtdInsPs and and neutral loss scans provided a relationship between signal and amounts of PtdInsPs. Precursor ion scans also of the ion of precursor ion scans of sn-1 MAG were than neutral loss scans of we used neutral loss scans for relative of PtdInsPs from biological due to ammonium ion PtdIns PtdInsP2 PtdInsP3 signal the peak of ammonium ion PtdInsPs to PtdInsPs. areas of ammonium ion or PtdInsPs were obtained ammonium or acid in in a new in the quantification of ammonium ion of PtdIns PtdInsP2 PtdInsP3 loss scans were and peak areas for PtdInsPs were used to the of ammonium ion The peak of PtdInsPs species to of the the relative of in a new The signal the peak of ammonium ion PtdInsPs to PtdInsPs. areas of ammonium ion or PtdInsPs were obtained ammonium or acid in loss scans were and peak areas for PtdInsPs were used to the of ammonium ion The peak of PtdInsPs species to of the the relative of PtdInsPs provided as major in CID as in the neutral loss to headgroups, have been performed to PtdInsPs. Clark J. Anderson K.E. V. Stephens L.R. Hawkins P.T. Quantification of PtdInsP3 species in cells and tissues by mass spectrometry.Nat. 8: PubMed Scopus Google also used a to and they used a neutral loss scan of to In the present study, a neutral loss scan of used to PtdInsP3 isomers due to ammonium ion neutral loss scans of and were used to and PtdIns from cells and A neutral loss scan provides the of phosphate groups in the inositol phosphates of fatty acyl compositions at the sn-1 and sn-2 positions could be CID for PtdInsPs be obtained to the structural of the lipid species J. Anderson K.E. V. Stephens L.R. Hawkins P.T. Quantification of PtdInsP3 species in cells and tissues by mass spectrometry.Nat. 8: PubMed Scopus Google Scholar). PtdInsPs in biological samples be from a neutral loss scan for structural of the and is and for all PtdInsPs be performed with of the method on PtdInsPs observed in biological the composition of fatty acyl in PtdInsPs could be identified with the neutral loss scan For example, from a neutral loss scan of be identified as fatty acyl composition could be of the or other precursor ion scans could the fatty acyl compositions at the sn-1 and sn-2 positions of PtdInsPs T. J. P. L. and relative of phosphoinositides by precursor ion on phosphate and 2015; PubMed Scopus Google Scholar). In our present study, precursor ion scans of sn-1 MAG were performed to sn-1 fatty acid of PtdInsPs in positive ion mode. Mass of PtdInsPs the fatty acyl compositions and of from neutral scan the PtdInsPs with sn-1 fatty acid samples were known to and fatty acids at the sn-1 of phospholipids A. A. acids of the and phospholipids of cells and Lipid Res. Full Text PDF Google Scholar). we precursor ion scans for and fatty acids, as as for and the precursor ion scan could be on the fatty acid compositions of PtdInsPs. The neutral loss scans for cells could and The generated from the neutral loss scan of for PtdIns are in The sn-1 and sn-2 fatty acids of PtdIns were with fatty acid compositions using the from precursor ion scans in PtdInsPs fatty acids or fatty acids were to as in Precursor ion scan of performed to PtdInsPs acid at the sn-1 in mass were generated for the for from neutral loss scan of shows in The peak from is from the peak be PtdIns species or be generated from the of as from For PtdInsPs were the PtdInsPs species the peak at as for and were as PtdIns and Precursor ion scans also of the ion of precursor ion scans of sn-1 MAG were than neutral loss scans of headgroups in positive ion mode. we used neutral loss scans for relative of PtdInsPs from biological were observed from neutral loss scan and their sn-1 fatty acyl compositions were PtdInsP2 and species with the fatty acyl compositions were observed at the as in and of precursor ion scans of to PtdInsPs fatty acids at sn-1 of from ammonium in the The areas in the the PtdInsPs and PtdInsPs were The of the of to and ammonium ion has been to Akt phosphorylation via of C. B. C. S. stimulation of a survival signaling for endothelial Biol. 2000; PubMed Scopus Google Scholar). analysis shows Akt phosphorylation by as in and PtdInsPs were to be of PtdInsPs in cells after stimulation are in PtdInsP3 after compared with the PtdInsP3 from The ion of precursor ion scans of sn-1 MAG were than neutral loss scans of we used neutral loss scans for the of PtdInsPs from biological for species of PtdInsPs from neutral scan analysis are in and the peak of all PtdInsPs observed are in of PtdInsPs in are in areas of all PtdInsPs observed are in PtdInsP3 were the most for compared with the PtdInsP3 from in after PtdIns PtdInsP2 levels in cells and were the PtdInsP3 after The major fatty acyl composition of PtdInsPs in and the amount of PtdInsPs of all observed PtdInsPs. shows the of the neutral loss scans of headgroups and precursor ion scans of sn-1 MAG is to quantitatively PtdInsPs with structural information of the sn-1 and sn-2 fatty acids, and ammonium ion adduction is to PtdInsPs. profiling of PtdInsPs in cells and tissues successfully performed with structural information of the sn-1 and sn-2 fatty acid were observed for of PtdInsPs in the samples we major fatty acid compositions of PtdInsPs were PtdInsPs for and these fatty acid compositions on the of cells and of different phosphorylation on the inositol could be with and of our