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A targeted mass spectrometric analysis of phosphatidylinositol phosphate species

2005; Elsevier BV; Volume: 46; Issue: 8 Linguagem: Inglês

10.1194/jlr.d500010-jlr200

1539-7262

Stephen Milne, Pavlina T. Ivanova, Dianne L. DeCamp, Robert C. Hsueh, H. Alex Brown,

Metabolism and Genetic Disorders

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3.This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate. The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate. Phosphatidylinositol phosphates are versatile signaling lipids involved in multiple cellular functions. They and their metabolites are important elements of cellular processes, including cell motility, polarity, apoptosis, oncogenesis, vesicle transport, membrane fusion, and calcium mobilization (1Le Blanc C. Mironneau C. Barbot C. Henaff M. Bondeva T. Wetzker R. Macrez N. Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate.Circ. Res. 2004; 95: 300-307Google Scholar, 2Menager C. Arimura N. Fukata Y. Kaibuchi K. PIP3 is involved in neuronal polarization and axon formation.J. Neurochem. 2004; 89: 109-118Google Scholar, 3Wang F. Herzmark P. Weiner O.D. Srinivasan S. Servant G. Bourne H.R. Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils.Nat. Cell Biol. 2002; 4: 513-518Google Scholar, 4Tian W. Laffafian I Dewitt S. Hallett M.B. Exclusion of exogenous phosphatidylinositol-3,4,5-trisphosphate from neutrophil-polarizing pseudopodia: stabilization of the uropod and cell polarity.EMBO Rep. 2003; 4: 982-988Google Scholar, 5Czech M.P. PIP2 and PIP3: complex roles at the cell surface.Cell. 2000; 100: 603-606Google Scholar). The production of phosphatidylinositol trisphosphate (PIP3) is particularly notable as it has been shown to be a regulator of many signaling processes and downstream molecules such as serine/threonine kinase and mammalian target of rapamycin. The broad role of PIP3 and related metabolites in signaling processes demands a worthy method for measuring its cellular activity. However, PIP3 is present in such low concentrations within the cell that direct measurement has been difficult. Traditional methods for phosphoinositide detection in cell extracts have included receptor displacement assays, metabolic labeling, and chromatographic separation of the radiolabeled products after deacylation (6Gray A. Van der Kaay J. Downes C.P. The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo.Biochem. J. 1999; 344: 929-936Google Scholar, 7Alter C.A. Wolf B. Identification of phosphatidylinositol 3,4,5-trisphosphate in pancreatic islets and insulin-secreting β-cells.Biochem. Biophys. Res. Commun. 1995; 208: 190-197Google Scholar, 8Nasuhoglu 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: 243-254Google Scholar). To identify phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and PIP3 compounds in total cell extracts, we strategically combined a selective extraction and mass spectrometric protocol. Lipidomic analysis permits the detection and identification of important membrane signaling molecules that were formerly difficult to fully characterize by other methods (9Milne, S. B., J. S. Forrester, P. T. Ivanova, M. D. Armstrong, and H. A. Brown. 2003. Multiplexed lipid arrays of anti-immunoglobulin M-induced changes in the glycerophospholipid composition of WEHI-231 cells. AfCS Res. Rep. Accessed June 3, 2005, at www.signaling-gateway.org/reports/v1/DA0011/DA0011.htm.Google Scholar, 10Forrester J.S. Milne S.B. Ivanova P.T. Brown H.A. Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction.Mol. Pharmacol. 2004; 65: 813-821Google Scholar, 11Ivanova P.T. Milne S.B. Forrester J.S. Brown H.A. Lipid arrays: new tools in the understanding of membrane dynamics and lipid signaling.Mol. Interv. 2004; 4: 84-94Google Scholar, 12Milne S.B. Ivanova P.T. Forrester J.S. Brown H.A. LIPIDOMICS: an analysis of cellular lipids by ESI-MS.Methods. 2005; In pressGoogle Scholar). This includes the assessment of changes in cellular levels of polyphosphoinositides. Traditionally, these species have been analyzed as a deacylated "pool" of 32P-labeled inositides by HPLC (7Alter C.A. Wolf B. Identification of phosphatidylinositol 3,4,5-trisphosphate in pancreatic islets and insulin-secreting β-cells.Biochem. Biophys. Res. Commun. 1995; 208: 190-197Google Scholar) or as intact compounds by TLC (13Hegelwald H. One-dimensional thin-layer chromatography of all known D-3 and D-4 isomers of phosphoinositides.Anal. Biochem. 1996; 242: 152-155Google Scholar). These procedures can result in qualitative estimates of total PIP and PIP2, but chemical identification of the species within the PIP/PIP2 pools (i.e., species distinguishable from one another by their fatty acyl chains) in the context of other changes in lipid species has been challenging. Electrospray ionization mass spectrometry has been applied to the analysis of polyphosphoinositides (i.e., PIP and PIP2) (14Wenk M.R. Lucast L. Paolo G. Di Romanelli A.J. Suchy S.F. Nussbaum R.L. Cline G.W. Shulman G.I. McMurray W. Camilli P. De Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.Nat. Biotechnol. 2003; 21: 813-817Google Scholar), but until now, the detection of PIP3 and the identification of its chemical composition have been elusive. Since its discovery in the late 1980s, PIP3 has been intensively studied because of its important role as a mediator in signal transduction and in many physiologically and pathologically important processes. Practically undetectable in unstimulated cells, PIP3 species formation increases rapidly in response to a variety of stimuli, including growth factors and oxidative stress. Reactive oxygen species function as mediators in cellular responses and are clinically important as factors for respiratory distress and other disorders. Although PIP3 generated as a result of growth factor stimulation is synthesized by the action of phosphatidylinositol-3-kinase on phosphatidylinositol 4,5-bisphosphate, exposure of the cells to oxidative stress activates a novel synthetic pathway via type I PIP kinase acting on phosphatidylinositol 3,4-bisphosphate (Fig. 1)(15Dobos G.J. Norgauer J. Eberle M. Schollmeyer P.J. Traynor-Kaplan A.E. C5a reduces formyl peptide-induced actin polymerization and phosphatidylinositol(3,4,5)phosphate formation, but not phosphatidylinositol(4,5)bisphosphate hydrolysis and superoxide production, in human neutrophils.J. Immunol. 1992; 149: 609-614Google Scholar, 16Qin S. Stadtman E.R. Chock P.B. Regulation of oxidative stress-induced calcium release by phosphatidylinositol-3-kinase and Bruton's tyrosine kinase in B cells.Proc. Natl. Acad. Sci. USA. 2000; 97: 7118-7123Google Scholar). After the production of PIP3, a host of signaling proteins with lipid recognition sites are localized to the plasma membrane and their signaling activity is enhanced or set in motion. Feedback loops that promote or control additional PIP3 production have been described for processes such as cell polarity and chemotaxis (17Rickert P. Weiner O.D. Wang F. Bourne H.R. Servant G. Leukocytes navigate by compass: roles of PI3Kγ and its lipid products.Trends Cell Biol. 2000; 10: 466-473Google Scholar). Clearly, PIP3 is an important signaling molecule whose activity is central to many cellular processes, including those involved in disease (18Cantley L.C. The phosphoinositide 3-kinase pathway.Science. 2002; 296: 1655-1657Google Scholar). The implication of PIP3-dependent signaling has received attention in cancer research, because deregulation of this signaling system [i.e., phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and SH2 (Src homology 2)-containing inositol phosphatase (SHIP)] facilitates tumor progression (19Hinchliffe K.A. Cellular signaling: stressing the importance of PIP3.Curr. Biol. 2001; 11: R371-R373Google Scholar, 20Leslie N.R. Downes C.P. PTEN function: how normal cells control it and tumour cells lose it.Biochem. J. 2004; 382: 1-11Google Scholar). The lack of adequate methods has not previously permitted investigators to determine whether distinct species of PIP3 might contribute to its broad signaling role or alter the formation of specialized proteolipidomic complexes that might be exploited pharmacologically. The importance of acyl chain specificity has been shown for diacylglycerol activation of protein kinase C (21Pettitt T.R. Martin A. Horton T. Liossis C. Lord J.M. Wakelam M.J. Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions.J. Biol. Chem. 1997; 272: 17354-17359Google Scholar, 22Marignani P.A. Epand R.M. Sebaldt R.J. Acyl chain dependence of diacylglycerol activation of protein kinase C activity in vitro.Biochem. Biophys. Res. Commun. 1996; 225: 469-473Google Scholar). Recent studies of the selectivity for phosphatidylinositol and phosphatidylcholine by phosphatidylinositol transfer protein suggests that head group chemistry is not the only determining factor in lipid binding affinity. Analysis of acyl chain length and saturation indicates that these are also critical factors in lipid binding and transfer (23Hunt A.N. Skippen A.J. Koster G. Postle A.D. Cockcroft S. Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein α.Biochim. Biophys. Acta. 2004; 1686: 50-60Google Scholar, 24Van Paridon P.A. Gadella T.W.J. Somerharju P.J. Wirtz K.W.A. Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain.Biochemistry. 1988; 27: 6208-6214Google Scholar). For example, the increased levels of lysophosphatidic acid (LPA) observed in ovarian cancer patients have been implicated as important markers of the disease. An increased quantity of unsaturated fatty acid LPA species is found in patients with late-stage ovarian cancer, thus making it a potential target for cancer therapy (25Umezu-Goto M. Tanyi J. J J. Lahad Liu S. Yu S. Lapushin R. Hasegawa Y. Lu Y. Trost R. Bevers T. et al.Lysophosphatidic acid production and action: validated target in cancer?.J. Cell. Biochem. 2004; 92: 1115-1140Google Scholar). Interestingly, a recent report suggests that the acyl chain length of PIP3 is an important determinant in the binding to the (CT-)SH2 domain of the p85α subunit of phosphatidylinositol 3-kinase (26Ching T-T. Lin H.P. Yang C.C. Oliveira M. Lu P.J. Chen C.S. Specific binding of the C-terminal Src homology 2 domain of the p85α subunit of phosphoinositide 3-kinase to phosphatidylinositol-3,4,5-trisphosphate.J. Biol. Chem. 2001; 276: 43932-43938Google Scholar). The catalytic properties of type II phosphoinositide 5-phosphatases, and also their biological functions, are not only factors of the localization (or their regulatory domain) but are highly dependent on the fatty acid and head group composition of the lipid substrate (27Schmid A.C. Wise H.M. Mitchell C.A. Nussbaum R. Woscholski R. Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation.FEBS Lett. 2004; 576: 9-13Google Scholar). RAW 264.7 cells are a macrophage-like, Abelson leukemia virus-transformed cell line derived from BALB/c mice. RAW 264.7 cells used by the Alliance for Cellular Signaling (AfCS) were obtained from the American Type Culture Collection (ATCC catalog number TIB-71, lot number 2263775), expanded, and stored in aliquots for use by AfCS laboratories. Stock vials of frozen AfCS cells were thawed and maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, and 2 mM l-glutamine at 37°C in a humidified atmosphere with 5% CO2. For routine maintenance in culture (passage), cells were seeded at a confluence of ∼10% (1 × 106 and 3 × 106 cells on 100 and 150 mm plates, respectively) and grown to a density of ∼80%. For ligand stimulation, cells were seeded at 4 × 106 cells on 60 mm plates. Before assay, the medium was changed to DMEM supplemented with 20 mM HEPES, 2 mM l-glutamine, and 0.1 mg/ml BSA. After the addition of C5a, LPA, zymosan, macrophage colony-stimulating factor (MCF), or control medium (growth medium), the plates were placed on ice and the media were aspirated at the appropriate time points. The cells were washed with 1.5 ml of ice-cold PBS solution and pelleted, and PBS was aspirated. For comparison, reactions were also terminated by the addition of cold 10% trichloroacetic acid, which gave similar results (data not shown). RAW 264.7 cells used by the LIPID MAPS consortium were obtained from the American Type Culture Collection (ATCC catalog number TIB-71, lot number 3002360). Likewise, a strain of RAW 264.7 cells commonly used at Vanderbilt Medical Center were originally obtained from the American Type Culture Collection (ATCC catalog number TIB-71). These additional sources for RAW 264.7 cell strains were all stored, grown and assayed according to the AfCS protocol described above. Stimulation with LPA, C5a, and zymosan was carried out in all cultured and primary murine macrophage cells. MCF experiments were performed on RAW 264.7 cells used by the LIPID MAPS consortium. Peritoneal macrophages were harvested from CD-1/ICR mice (standard outbred strain). The lavage fluid from the mice was centrifuged to a pellet and resuspended in α-MEM containing 10% fetal calf serum and penicillin at 2 ml/mouse. The cells were plated onto 60 mm plates at 6 ml (three mice)/plate, incubated for 2 h, and then washed with warm PBS to remove the lymphocytes. The macrophages were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, and 2 mM l-glutamine at 37°C in a humidified atmosphere with 5% CO2 overnight. Ice-cold 1:1 CHCl3/CH3OH (400 μl) was added to each pellet and vortexed for 1 min, or until thoroughly mixed. Samples were centrifuged at 9,000 rpm for 5 min at 4°C, supernatant decanted, and discarded. To the remainder of the cell pellet, 200 μl of 2:1 CHCl3/CH3OH containing 0.25% 12 N HCl was added. Samples were vortexed for 5 min and then pulse spun. To the supernatant, 40 μl of 1 N HCl was added and vortexed for 15 s. Samples were centrifuged to separate the phases. The solvent from the collected lower layer was evaporated in a vacuum centrifuge (Labconco CentriVap Concentrator, Kansas City, MO), and lipid film was rapidly redissolved in 55 μl of 1:1:0.3 CHCl3/CH3OH/H2O. Before analysis, 5 μl of 300 mM piperidine (28Lytle C.A. Gan Y.D. White D.C. Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection.J. Microbiol. Methods. 2000; 41: 227-234Google Scholar) was added, and the sample was vortexed and pulse spun. Mass spectral analysis was performed on either an MDS SCIEX 4000 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA) or a Finnigan TSQ Quantum triple quadrupole mass spectrometer (ThermoFinnigan, San Jose, CA). Both instruments were equipped with a Harvard Apparatus syringe pump and an electrospray source. Samples were analyzed at an infusion rate of 10 μl/min in negative ionization mode over the range of m/z 400–1,200. Because of their very low abundance and detection only after stimulation, PIP3 species cannot be quantitated or identified by traditional full scan or precursor ion scan (m/z 481) techniques. If much larger sample sizes were available, quantitation might be possible by precursor ion scan with the use of an internal standard such as 16:0 PIP3 (phospholipids are presented with the class abbreviation preceded by xx:y, where xx is the total carbon atoms in the fatty acid chains and y is the number of double bonds). Instead, qualitative identification of the individual phosphatidylinositol phosphates present in the total lipid extracts was accomplished by electrospray ionization tandem mass spectrometry with a collision energy of 50 eV. Peaks corresponding to known PIP3s were fragmented and inspected manually for the presence of the identification peaks (Table 1). A confirmed identification was achieved when key fragmentation peaks were greater than three times the signal-to-noise ratio. The lower limit of detection using this method was found to be <9 pmol/ml for 38:4 PIP3 (Avanti Polar Lipids, Alabaster, AL). Data were collected with either the Analyst software package (Applied Biosystems) or the Xcalibur software package (ThermoFinnigan).TABLE 1Key fragments from the eight PIP3 species identified by collision-induced dissociation tandem mass spectrometryPIP3m/zFatty Acid 1 (m/z)Fatty Acid 2 (m/z)LPA 1 (m/z)LPA 2 (m/z)LPA-H2O (m/z)LPA-H2O (m/z)32:11,04716:0 (255)16:1 (253)16:0 (409)16:0 (391)32:01,04916:0 (255)16:0 (255)16:0 (409)16:0 (409)16:0 (391)16:0 (391)34:11,07516:0 (255)18:1 (281)18:0 (437)16:0 (391)18:1 (417)18:0 (283)16:1 (253)18:0 (419)16:1 (389)36:21,10118:1 (281)18:1 (281)18:1 (435)18:1 (435)18:1 (417)18:1 (417)36:11,10318:0 (283)18:1 (281)18:0 (419)18:1 (417)38:41,12518:1 (281)20:3 (305)18:1 (435)20:3 (441)38:31,12718:2 (279)18:2 (433)PIP330332133938340141946348149932:1+++++++++32:0+++++++++34:1++++++++36:2++++++++36:1++++38:4+++++38:3+++++LPA, lysophosphatidic acid; PIP3, phosphatidylinositol trisphosphate. The m/z 303, 321, and 339 peaks are inositol bisphosphate fragments [produced from phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2), or PIP3 species]; the m/z 383, 401, and 419 peaks are inositol trisphosphate fragments (produced from PIP2 or PIP3 species); and the m/z 463, 481, and 499 peaks are inositol-1,3,4,5-tetrakisphosphate fragments (only produced from PIP3 species). + indicates that this fragment was observed during collision-induced dissociation tandem mass spectrometry analysis. Open table in a new tab LPA, lysophosphatidic acid; PIP3, phosphatidylinositol trisphosphate. The m/z 303, 321, and 339 peaks are inositol bisphosphate fragments [produced from phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2), or PIP3 species]; the m/z 383, 401, and 419 peaks are inositol trisphosphate fragments (produced from PIP2 or PIP3 species); and the m/z 463, 481, and 499 peaks are inositol-1,3,4,5-tetrakisphosphate fragments (only produced from PIP3 species). + indicates that this fragment was observed during collision-induced dissociation tandem mass spectrometry analysis. PIP3s constitute only ∼0.25% of the inositol lipids in eukaryotic cell membranes. To minimize the interference of other phospholipids during analysis, a selective two-step extraction was used. First, the cell material was extracted with neutral solvents [somewhat similar to steps described previously (29Gray A. Olsson H. Batty I.H. Priganica L. Downes C.P. Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts.Anal. Biochem. 2003; 313: 234-245Google Scholar)], and the resulting pellet was extracted with acidified solvents for quantitative recovery of polyphosphoinositides. The majority of noninositol phospholipids were extracted with neutral solvents, and no PIP2 or PIP3 species were detected in this extract. Using electrospray ionization tandem mass spectrometry, we have surmounted many obstacles and can now identify a plethora of intact polyphosphoinositides, including PIP3. Collision-induced dissociation of the peaks of interest yielded fragmentation patterns that were used to unambiguously identify the lipids present at a particular m/z value (Fig. 2)(30Hsu F-F. Turk J. Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study.J. Am. Soc. Mass Spectrom. 2000; 11: 986-999Google Scholar). The molecular weights of two PIP3 compounds detected coincided with PIP2 compounds (32:0 PIP3/38:2 PIP2 at m/z 1,049, and 32:1 PIP3/38:3 PIP2 at m/z 1,047). However, this in no way interfered with the identification process, because key fragments attributable only to PIP3 species were used to classify these compounds. A list of the major fragments for the eight PIP3 species observed to date is summarized in Table 1. A typical fragmentation pattern from a PIP3 compound includes the two fatty acids (fatty acids 1 and 2), LPAs (LPA 1 and LPA 2) as well as their dehydration products (LPA-H2O), and inositol-1,3,4,5-tetrakisphosphate fragments. As shown in Fig. 3A, agonist (zymosan) challenge of RAW 264.7 cells led to increased levels of a wide variety of PIP and PIP2 species compared with basal levels. All of the polyphosphoinositides identified in basal RAW 264.7 cells were also detected in primary macrophages. In basal samples, the two systems differed in the levels of 36:2, 36:1, and 38:4 series phosphatidylinositol, PIP, and PIP2 compounds. Primary macrophages have conspicuously higher levels of 38:4 series phosphoinositides compared with RAW 264.7 cells (Fig. 3B).Fig. 3Electrospray ionization mass spectrometry for PIP and phosphatidylinositol bisphosphate (PIP2) compounds in basal and ligand-stimulated cells. A: When treated with 50 μg/ml zymosan for 15 min, RAW 264.7 cells showed a dramatic increase in PIP and PIP2 levels compared with basal conditions. B: Unstimulated primary murine macrophages and RAW 264.7 phosphatidylinositol phosphate compositions are markedly different.View Large Image Figure ViewerDownload (PPT) As shown in Table 2, different PIP3 species were produced as a result of distinct ligand stimulation, suggesting differential mechanisms of enzyme activation or selective substrate availability triggered by the specific agonist. Although stimulation with 2.5 μM LPA elicited 32:0 and 36:2 PIP3 species, treatment with 10 nM C5a, 400 pM MCF, or 125 μg/ml zymosan produced a wider range of molecular species. The stimulation times were selected by identifying the optimal time points for maximum PIP3 activity measured by the peak of serine/threonine kinase phosphorylation and Ca2+ release (data not shown). We observed some differences between the molecular species generated in RAW 264.7 cells and primary macrophages for a given ligand, but in general there was significant conservation in the PIP3 species generated. Interestingly, no inter-cell-type differences in PIP3 content were observed among the three different sources of RAW 264.7 cells after LPA or C5a stimulation. This suggests that primary macrophage lipid signaling pathways are well conserved in the various RAW 264.7 cell strains available.TABLE 2PIP3 compounds detected after LPA, C5a, zymosan, or MCF stimulationLigandConcentrationTimeDetected PIP3 SpeciesPrimary murine macrophagesLPA 2.5 μM45 s1,049, 1,075, 1,101, 1,125 (32:0, 34:1, 36:2, 38:4)C5a 10 nM45 s1,101 (36:2)Zymosan 125 μg/ml10 min1,075, 1,101, 1,125, 1,127 (34:1, 36:2, 38:4, 38:3)RAW 264.7 cellsLPA 2.5 μM30 s1,049, 1,101 (32:0, 36:2)C5a 10 nM45 s1,049, 1,075, 1,101, 1,103, 1,125 (32:0, 34:1, 36:2, 36:1, 38:4)Zymosan 125 μg/ml10 min1,049, 1,075, 1,101, 1,103 (32:0, 34:1, 36:2, 36:1)MCF 400 pM3 min1,075, 1,101, 1,103, 1,125, 1,127 (34:1, 36:2, 36:1, 38:4, 38:3)Parallel experiments were performed with different strains of RAW 264.7 cell lines obtained from various sources, including the Alliance for Cellular Signaling and LIPID MAPS projects. Experiments with LPA, C5a, and zymosan were performed with all cell lines. Macrophage colony-stimulating factor (MCF) stimulation was carried out in LIPID MAPS RAW 264.7 cells only. Open table in a new tab Parallel experiments were performed with different strains of RAW 264.7 cell lines obtained from various sources, including the Alliance for Cellular Signaling and LIPID MAPS projects. Experiments with LPA, C5a, and zymosan were performed with all cell lines. Macrophage colony-stimulating factor (MCF) stimulation was carried out in LIPID MAPS RAW 264.7 cells only. Using lipidomic analysis, we unequivocally identified ∼75 phosphatidylinositol and phosphatidylinositol phosphate compounds (Table 3) according to their acyl composition. We have observed that the phosphatidylinositol phosphate "fingerprint" can vary greatly between different cell types. Additionally, results suggest that the PIP3 species formed after cell stimulation are ligand-dependent. This finding implies that PIP3 may not function as a single entity in cells. Rather, there appear to be eight or more species in the macrophage alone, functioning as a lipid second messenger. Whether these distinct species have differential binding preferences to downstream effector molecules remains to be determined. However, this virtual lipid array of signaling polyphosphoinositides allows us to begin the molecular dissection of various cellular outcomes regulated by differential molecular species of PIP3 and to establish substrate-product relationships between essential signaling lipid pools that are associated with a specific cell surface receptor in a way that has not previously been possible.TABLE 3Summary table of acyl chain composition and m/z values of phosphatidylinositol (PI), PIP, PIP2, and PIP3 species identified in RAW 264.7 macrophage cells32:032:132:234:034:134:236:036:136:236:336:438:038:138:238:338:438:540:440:540:6PI809807805837835833865863861859857893891889887885883913911909PIP889887885917915913945943941939937973971969967965963993991989PIP29699679659979959931,0251,0231,0211,0191,0171,0531,0511,0491,0471,0451,0431,0731,0711,069PIP31,0491,0471,0451,0771,0751,0731,1051,1031,1011,0991,0971,1331,1311,1291,1271,1251,1231,1531,1511,149Boldface values indicate identified peaks. Open table in a new tab Boldface values indicate identified peaks. This work was supported by contributions from public and private sources, including the National Institute of General Medical Sciences Glue Grant Initiative AfCS (U54 GM062114), National Institutes of Health R01 GM-58516, and LIPID MAPS (U54 GM069338) http://www.lipidmaps.org. A complete listing of AfCS sponsors can be found at http://www.signaling-gateway.org/aboutus/sponsors.html. The authors thank Andrew Goodman, Michelle Armstrong, and Johnna Goodman for excellent technical and administrative support and Gil Sambrano at the University of California, San Francisco, for helpful discussions. The peritoneal macrophages were provided by Dr. Carol Rouzer (Department of Biochemistry, Vanderbilt University Medical Center).