A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Purification and Analysis of Mycobacterial Phosphatidylinositol Mannosides, Lipomannan, and Lipoarabinomannan.

Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.

Fatty-acyl chain profiles of cellular phosphoinositides.

Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2 pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in <20s of both PIP2 and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.

Detection and manipulation of phosphoinositides.

Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of PIs in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the PIs and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular PIs and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides.

Highly purified mycobacterial phosphatidylinositol mannosides drive cell-mediated responses and activate NKT cells in cattle.

Mycobacterial lipids play an important role in the modulation of the immune response upon contact with the host. Using novel methods, we have isolated highly purified phosphatidylinositol mannoside (PIM) molecules (phosphatidylinositol dimannoside [PIM2], acylphosphatidylinositol dimannoside [AcPIM2], diacyl-phosphatidylinositol dimannoside [Ac2PIM2], acylphosphatidylinositol hexamannoside [AcPIM6], and diacylphosphatidylinositol hexamannoside [Ac2PIM6]) from virulent Mycobacterium tuberculosis to assess their potential to stimulate peripheral blood mononuclear cell (PBMC) responses in Mycobacterium bovis-infected cattle. Of these molecules, one (AcPIM6) induced significant levels of gamma interferon (IFN-γ) in bovine PBMCs. Three PIM molecules (AcPIM6, Ac2PIM2, and Ac2PIM6) were shown to drive significant proliferation in bovine PBMCs. AcPIM6 was subsequently used to phenotype the proliferating cells by flow cytometry. This analysis demonstrated that AcPIM6 was predominantly recognized by CD3(+) CD335(+) NKT cells. In conclusion, we have identified PIM lipid molecules that interact with bovine lymphocyte populations, and these lipids may be useful as future subunit vaccines or diagnostic reagents. Further, these data demonstrate, for the first time, lipid-specific NKT activation in cattle.

Mass spectrometric analyses of phospholipids in the S334ter-3 rat model of retinal degeneration.

PURPOSE: The purpose of this study was to profile the endogenous phospholipid species in the retinal tissue of the S334ter-3 rat model of retinal degeneration. Retinal tissue was collected from S334ter-3 rats at postnatal day (P) 20, P30, and P60, while control retinal samples were collected from Sprague-Dawley (SD) rats at the same time points for comparison. METHODS: Lipids were extracted using the Bligh and Dyer method, and resuspended in an acetonitrile/isopropanol (1:1) solution. For lipid analyses, a positive ion-mode precursor ion scan (PIS) was used for phosphatidylcholine (PC; product m/z of 184), a negative ion-mode neutral loss scan (NLS) was used for phosphatidylserine (PS; product m/z of 87.1), and a negative ion-mode PIS was used for phosphatidylinositol (PI; product m/z of 241) and phosphatidylethanolamine (PE; product m/z of 196); the analyses were carried out using a TSQ Quantum Access Max mass spectrometer. The samples were directly infused with a Triversa Nanomate using 1.6 kV and 0.4 psi of pressure for the positive ion mode, and 1.3 kV and 0.6 psi of pressure for the negative ion mode, and scanned for 2 min between 200 m/z and 1000 m/z. Ratiometric quantification was performed using quantitative standards for each lipid class. RESULTS: The comparative profiles of PC, PE, PS, and PI between S334ter-3 and control rats showed that there were several lipid species common to both groups, as well as several that were unique to the S334ter-3 group and vice versa. CONCLUSIONS: It was found that the proportions of PC and PS were higher in the control retina compared to S334ter-3, and that the proportions of PE and PI were higher in the S334ter-3 retina compared to control.

Phosphoinositide 3-kinase assay in breast cancer cell extracts.

Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate multiple biological functions such as cell growth, proliferation, migration, and survival. Class I PI3Ks consist of four kinases isoforms. Over the past years many studies have documented that each isoform of PI3K plays specific biological functions in different cell types. Accumulating evidence indicates that activation of PI3K signaling is deregulated in human disease, including cancer. A major pharmaceutical effort has gone into developing PI3K inhibitors that hit multiple or individual PI3K isoforms, which are currently used in early and late-phase clinical trials. In this chapter we describe an in vitro PI3K assay that may be helpful in verifying which tumor cells have increased PI3K activity and thus may be targeted with inhibitors of PI3K.

The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT) compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS) showed that these molecules included 14 phosphatidylinositols (PIs), 3 phosphatidylethanolamines (PEs) and 3 phosphatidic acids (PAs). Among the PIs, the expression of PI(18:0/18:1), PI(18:0/20:3) and PI(18:0/20:2) were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA). The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

[Chemical constituents from aerial part of Rumex patientia].

OBJECTIVE: To study the chemical constituents from aerial part of Rumex patientia. METHODS: The compounds were isolated and purified by silica gels and polyamide column chromatography. Their structures were elucidated by physicochemical and spectroscopic evidences. RESULTS: Twelve compounds were identified as: chrysophanol (1), chrysophanol-8-O-beta-D-glucopyranoside (2), physcion (3), emodin(4), emodin-8-O-beta-D-glucopyranoside (5), maackiain (6), maackiain-3-O-beta-D-glucopyranoside (7), quercetin-3-O-beta-D-glucopyranoside (8), quercetin-3-O-beta-D-glucuronide(9), 2-O-methylinositol (10), torachrysone-8-O-beta-D-glucopyranoside (11) and nepodin-8-O-beta-D-glucopyranoside (12). CONCLUSION: Compounds 6, 7, 10 are isolated from this genus for the first time, and compound 9 is isolated from this plant for the first time.

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis.

Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system. PRINCIPAL FINDINGS: We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect. CONCLUSIONS: These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.

Devising powerful genetics, biochemical and structural tools in the functional analysis of phosphatidylinositol transfer proteins (PITPs) across diverse species.

The minor cellular lipid phosphoinositides represents key regulators of diverse intracellular processes such as signal transduction at membrane-cytosol interface, regulation of membrane trafficking, cytoskeleton organization, nuclear events and the permeability, and transport functions of the membrane. The heterogeneous subcellular localization of phosphoinositides and their multiple and co-operative membrane-protein recognition mechanisms contribute to a "coincidence detection code" for the membrane-cytosol interactions in eukaryotic signaling networks. Such a "coincidence detection code" relies on the fine coordination of the broader lipid metabolism and organization, and their coupling to dedicated physiological processes. The phosphatidylinositol transfer proteins (PITPs) play a key regulatory role, essentially as "coincidence detectors" or "nanoreactors" in this "signal detection code" that spatially and temporally coordinate the diverse aspects of lipid metabolome with phosphoinositide signaling to effect various cellular functions. The integral role of PITPs in the highly conserved eukaryotic signal transduction strategy is amply demonstrated by the mammalian diseases associated with the derangements in the function of these proteins, to stress response and developmental regulation in plants, to fungal dimorphism and pathogenicity, to membrane trafficking in yeast and higher eukaryotes. The study of PITPs is fundamental to understanding of how the phosphoinositide signal transduction network is regulated and integrated to the larger lipid metabolome in diverse cellular processes. To comprehend how the PITPs integrate phosphoinositide signaling to broader lipid metabolome in diverse cellular processes, it is necessary to devise methods that can correlate the biochemical properties of these non-enzymatic proteins to biologically relevant functional insights. In this chapter, we present combinatorial approaches that primarily employ genetics and structural tools to assess the functional role of PITPs in yeast, plant and mammalian systems. An elaborate discussion on the various genetic models devised for interpreting the functional role of PITPs in relation to their operational assays has been included. We also describe the structural and biophysical methods that have advanced our understanding of how these proteins operate as "nanoreactor" molecules.

The role of inositol and the principles of labelling, extraction, and analysis of inositides in mammalian cells.

Inositides have an important impact on diverse areas of cellular regulation. However, since this area has grown exponentially from the mid 1980s onwards, many workers find themselves relatively new to the field. In this chapter, we establish a broad foundation for the rest of the book by covering some important principles of inositide methodologies. The focus is especially directed to those methods or aspects of methodology not covered in detail in other chapters. This includes the often neglected influence of the inositide precursor, inositol, and important background information relating to the labelling and extraction of inositides from cells and tissues. This introductory section also gives a "birds eye" view of important methods and protocols found within this volume and hopefully acts as a touchstone to assess which of the methodologies described within this book is most appropriate for your particular study(ies) of inositides.

Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC.

The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P(2), PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3)) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.

Phosphoinositide analysis by liquid chromatography-mass spectrometry.

The phosphoinositides are a highly dynamic group of molecules implicated in many cellular control processes; however, the analysis of many of these structures has proven very difficult and time-consuming, with limited sensitivity and/or discrimination. Recent developments in LCMS now provide the prospect of routine structural and quantitative analysis of all the known phosphoinositides (and possibly some as yet unidentified structures) at high sensitivity in any biological sample. The procedures described here give very high extraction recovery from a variety of biological matrices and enable chromatographic resolution of most phosphoinositides as their native structures. When coupled with the accurate mass and fragmentation capabilities of an MS, full structural and isomeric identification can be achieved.

Methods to assess changes in the pattern of nuclear phosphoinositides.

Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signalling system that is regulated in response to a variety of extra and intra-cellular cues (Curr Opin Genet Dev 14:196-202, 2004). Phosphoinositides and the enzymes that synthesize them are found in many different sub-cellular compartments including the nuclear matrix, heterochromatin, and sites of active RNA splicing, suggesting that phosphoinositides may regulate specific functions within the nuclear compartment (Nat Rev Mol Cell Biol 4:349-360, 2003; Curr Top Microbiol Immunol 282:177-206, 2004; Cell Mol Life Sci 61:1143-1156, 2004). The existence of distinct sub-cellular pools has led to the challenging task of understanding how the different pools are regulated and how changes in the mass of lipids within the nucleus can modulate nuclear specific pathways. Here we describe methods to determine how enzymatic activities that modulate nuclear phosphoinositides are changed in response to extracellular stimuli.

Brain phosphoinositide extraction, fractionation, and analysis by MALDI-TOF MS.

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) can provide rapid, sensitive determinations of lipids from small tissue samples in both single determinations and automated high-throughput assays. MALDI-TOF MS is a sensitive, high-throughput technique for the determination of lipids such as the phosphoinositides, PtdIns (phosphatidylinositol), PIP (phosphatidylinositol-4-phosphate, and PIP2 (phosphatidylinositol-4,5-bisphosphate), but in crude extracts the signals are weak or not observed due in large part to ion suppression by phosphatidylcholine and other cationic lipids. A rapid separation step using a small column of a strong cation exchange (SCX) gel can be utilized easily and effectively to adsorb or capture cationic lipids from chloroform/methanol lipid extracts and provide substantially improved signals for the phosphoinositides. In this review, we describe the use of fractionation of a crude lipid extracts using cation exchange columns in conjunction with MALDI-TOF MS and appropriate internal standards to quantify the levels of phosphoinositides in small mammalian brain samples.

Composition analysis of positional isomers of phosphatidylinositol by high-performance liquid chromatography.

An HPLC-based method has been developed for composition analysis of six positional isomers of phosphatidylinositol (PI), of which the phosphatidyl group was connected to different positions of the myo-inositol moiety. The method employed a combination of two types of HPLC analyses. One was direct separation of the six PI isomers into four peaks of 1(3)-PI, 2-PI, 4(6)-PI and 5-PI on a normal-phase silica gel column. The second method was for the separations of 1-PI from 3-PI and 4-PI from 6-PI, which were not separable on the normal-phase column. This method involved conversion of PI isomers into pentakis-(R)-1-phenylethylcarbamate (PEC) derivatives, which were separated on a reversed-phase column. Using the established method, positional specificity of several engineered phospholipases D in enzymatic synthesis of PI from myo-inositol and phosphatidylcholine was investigated. This was performed by analyzing the isomeric composition of PIs synthesized by the mutant enzymes. Among five mutant enzymes tested, two showed strong specificity to 1-OH, one showed moderate preference to 1-OH, one preferred 3-OH, and one showed broad specificity towards 1-, 3-, 4- and 6-OH.

Lipidomic analysis of phospholipids and related structures by liquid chromatography-mass spectrometry.

Lipids are highly dynamic molecules with many different roles ranging from structural to signaling. Alterations in particular lipid levels change cellular behavior. In humans, inappropriate changes may manifest as diseases such as Alzheimer's, atherosclerosis, diabetes, obesity, and even cancer. This has led to a great interest in monitoring lipid changes both during normal cellular processes and in disease situations. With the advent of rapid mass spectrometry, it has become possible to analyze many lipids within a single sample with unsurpassed sensitivity and detail. Coupling this with liquid chromatography potentially enables the complete profiling of all the phospholipids, both major and minor, from a single sample within a single analysis run.