Molecular species analysis of lipids by metastable ion mass spectrometry

A convenient universal and fast mass spectrometrical method designed for the molecular species analysis of natural lipids is described. In contrast to the commonly employed procedures the method does not require chemical or enzymatic treatment and does not include chromatographic steps. The method relies on the recognition of ions characteristic of individual molecular species in the mass spectrum of a particular lipid fraction, that is accomplished on the basis of metastable ion spectra. The efficiency of this approach is demonstrated with a variety of natural lipids: triglycerides, glycerophospholipids, sphingomyelin and ornithinolipids. The advantages and limitations of the method as well as possible further developments are discussed.     

Structural analysis of mycolic acids from phenol-degrading strain of Rhodococcus erythropolis by liquid chromatography–tandem mass spectrometry

We used reversed phase liquid chromatography?Celectrospray ionization tandem mass spectrometry for direct analysis of mycolic acids (MAs) from four different cultivations of Rhodococcus erythropolis. This technique enabled us to identify and quantify the specific molecular species of MAs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. We identified a total of 60 molecular species of MAs by means of LC/MS. In collision-induced dissociation tandem mass spectrometry, the [M-H]^? ions eliminated two residues, i.e., meroaldehyde and carboxylate anions containing ??-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths and number of double bond(s). Two strains, i.e., R. erythropolis CCM 2595 and genetically modified strain CCM 2595 pSRK 21 phe were cultivated on two different substrates (phenol and phenol with addition of humic acids as a sole carbon source). The addition of humic acids showed that there is a marked increase of unsaturated mycolic acids, mostly in the range of 20?C100?%. This effect is more pronounced in the R. erythropolis CCM 2595 strain.     

HPLC/HR-MS-Based Metabolite Profiling and Chemometrics: A Powerful Approach to Identify Bioactive Compounds from Abarema cochliacarpos

Despite its importance as a medicinal plant, there is a lack of studies that assessed the chemical composition of A. cochliacarpos extracts. Herein, we used a metabolite profiling approach and chemometrics as a powerful strategy to correlate the chemical composition with the antioxidant activity of A. cochliacarpos extracts. Extracts obtained with ethyl acetate showed greater antioxidant activity and higher total phenolic content than extracts obtained with hexane. The chemical composition was assessed by HPLC/HR-MS and it encompassed fatty alcohols, terpenoids, phenolic derivatives, lipids, carotenoid-like compounds, alkaloids, flavonoids, polyketides, and glycerophospholipids. Chemometrics successfully differentiated not only the chemical composition of extracts in response to the nature of the extraction solvent and the botanical part used during extraction but also it allowed us to associate the chemical composition with the antioxidant activity of the extracts, which might be particularly helpful for drug discovery and development programs.     

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.     

An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.     

Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

Herein we describe a rapid, simple, and reliable method for the quantitative analysis and molecular species fingerprinting of triacylglycerides (TAG) directly from chloroform extracts of biological samples. Previous attempts at direct TAG quantitation by positive-ion electrospray ionization mass spectrometry (ESI/MS) were confounded by the presence of overlapping peaks from choline glycerophospholipids requiring chromatographic separation of lipid extracts prior to ESI/MS analyses. By exploiting the rapid loss of phosphocholine from choline glycerophospholipids, in conjunction with neutral-loss scanning for individual fatty acids, overlapping peaks in the ESI mass spectrum were deconvoluted generating a detailed molecular species fingerprint of individual TAG molecular species directly from chloroform extracts of biological samples. This method readily detects as little as 0.1 pmol of each TAG molecular species from chloroform extracts and is linear over a 1000-fold dynamic range. The sensitivity of individual TAG molecular species to ESI/MS/MS analyses correlated with the unsaturation index and inversely correlated with total aliphatic chain length of TAG. An algorithm was developed which identifies sensitivity factors, thereby allowing the rapid quantitation and molecular species fingerprinting of TAG molecular species directly from chloroform extracts of biological samples.     

Apoptosis-associated changes in the glycerophospholipid composition of hematopoietic progenitor cells monitored by 31P NMR spectroscopy and MALDI-TOF mass spectrometry

Apoptosis, or programmed cell death, plays an important role in development and in tissue homeostasis and is assumed to be accompanied by changes in the composition of cellular glycerophospholipids (GPL). We have applied a combination of ³¹P nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to the analysis of organic extracts of hematopoietic progenitor cells undergoing the physiologically relevant process of apoptosis following growth factor withdrawal. The combined application of these methods enables the quantitative analysis of all glycerophospholipid classes and reveals changes in the acyl chain compositions from crude cell extracts. Using these techniques, an increase in the ratio of ether-linked glycerophospholipids to diacyl-glycerophospholipids during apoptosis was observed. The relative decrease in the membrane diacyl-phosphatidylcholine (PC) levels was found to correlate with increased concentrations of the corresponding lysophosphatidylcholine (LPC) present in the medium.     

Human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Simple procedures for the rapid cleavage of ester lipids and for the large-scale isolation from brain of cerebroside sulfate

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry

Previous studies have shown that MALDI-imaging mass spectrometry (IMS) can be used to visualize the distribution of various biomolecules, especially lipids, in the cells and tissues. In this study, we report the cell-selective distribution of PUFA-containing glycerophospholipids (GPLs) in the mouse brain. We established a practical experimental procedure for the IMS of GPLs. We demonstrated that optimization of the composition of the matrix solution and spectrum normalization to the total ion current (TIC) is critical. Using our procedure, we simultaneously differentiated and visualized the localizations of specific molecular species of GPLs in mouse brain sections. The results showed that PUFA-containing phosphatidylcholines (PCs) were distributed in a cell-selective manner: arachidonic acid- and docosahexaenoic acid-containing PCs were seen in the hippocampal neurons and cerebellar Purkinje cells, respectively. Furthermore, these characteristic localizations of PUFA-PCs were formed during neuronal maturation. The phenomenon of brain cell-selective production of specific PUFA-GPLs will help elucidate the potential physiological functions of PUFAs in specific brain regions.     

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic “fingerprints” are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.     

Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is an established tool for the analysis of proteins, whereas it gained by far less interest in the field of lipid analysis. This method works well with phospholipids as well as organic cell extracts and provides high sensitivity and reproducibility. The aim of the present paper is to extend our previous studies to the analysis of lysophospholipids and phospholipid mixtures. To study the suitability of MALDI-TOF mass spectrometry for the analysis of lysophospholipids, different phospholipids like phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and phosphatidylinositol as well as their mixtures were digested with phospholipase A(2). Positive and negative ion mass spectra of all phospholipids before and after digestion were recorded. In all these cases, the molecular ions of the expected digestion products could be detected and only a very small extent of further fragmentation was observed. On the other hand, spectra of phospholipid mixtures containing phosphatidylcholine were strongly dominated by phosphatidylcholine and lysophosphatidylcholine signals, which prevented the detection of further phospholipids even if those lipids were present in comparable amounts. This is of paramount interest for the analysis of tissue and cell extracts.     

LIPID arrays: new tools in the understanding of membrane dynamics and lipid signaling

Phospholipids are the structural building blocks of the membrane bilayer, which retains and regulates intra-cellular content. In addition to creating a protective barrier around the cell, lipids modulate membrane trafficking and are themselves precursors of important intracellular signaling molecules. Identification and quantification of these molecular species is essential for a more complete understanding of cell signaling pathways, and more reliable and sensitive methods are needed for determining membrane phospholipid content. Recent improvements in electrospray ionization mass spectrometry have made possible the direct identification of more than 400 phospholipid species from biological extracts of a single cell type. Changes in the cellular concentration of diverse lipids can be determined by analysis of the mass spectra by statistical algorithms. In the future, lipid arrays will be integrated with other high-throughput profiling technologies, and computational lipidomics will expand our understanding of the molecular basis of cellular processes and diseases.     

Lipidomics: an analysis of cellular lipids by ESI-MS

Recognition of the importance of lipid signaling in cellular function has led to rapid progress in the technology of lipid analysis. Measurements of lipid species changes are central to defining the networks of cell signaling (e.g., receptor activation by hormones or drugs) and lipids are involved in many biochemical and pathological processes. During the last several years our laboratory has focused on developing efficient methods for extraction of glycerophospholipids from biological systems and their detection and identification by mass spectrometry. We analyze phospholipid changes in mammalian cells as a result of a defined ligand stimulation strategy that supports the research questions of the consortium. The improvement of mass spectrometry techniques for phospholipid analysis combined with sophisticated computational methods developed in our group has facilitated simultaneous analysis of hundreds of phospholipid species in mammalian cells. This information is presented as Lipid Arrays (or more precisely as virtual arrays) and allows identification of temporal changes in membrane phospholipid species between two contrasting biological conditions (e.g., unstimulated basal vs. stimulated or as a contrast between normal and disease stages). Using the lipidomics approach, we are able to identify approximately 450 phospholipid species from total membrane extracts and qualitatively measure pattern response changes initiated by cell surface receptors. As such, this approach facilitates the elucidation of the metabolic changes induced by a perturbation in the cell and recognition of patterns of signaling.     

Rapid characterization of artemether and its in vitro metabolites on incubation with bovine hemoglobin, rat blood and dog blood by capillary gas chromatography–chemical ionization mass spectrometry

A fast and sensitive analytical method was developed to characterize artemether and its metabolites in small amounts in body fluids. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide, separated on an optimized capillary gas chromatographic system and identified by chemical ionization mass spectrometry by using ammonia as reagent gas. The analytical assay is demonstrated on samples extracted from bovine hemoglobin, rat blood and dog blood. Full mass spectra of artemether and three metabolites were obtained at a level of 1·10⁻⁶ g/ml.     

Qualitative characterization of the rat liver mitochondrial lipidome using LC–MS profiling and high energy collisional dissociation (HCD) all ion fragmentation

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC–MS method that is able to monitor the major lipid classes found in biospecimens (i.e. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion high energy collisional dissociation fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC–MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC–MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.     

Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection

Phosphatidic acid (PA) and lysophosphatidic acid (LPA) are lipids that regulate cellular processes. PA stimulates kinases and may play a role in exocytosis and membrane fusion. LPA can induce cell proliferation, platelet aggregation, and microfilament formation. Due to the growing interest in these lipids, rapid purification and quantification of these lipids is desirable. We now describe a method that utilizes one HPLC run to separate trace amounts of PA and LPA from large amounts of lipids found in cellular extracts. A two-pump HPLC with a solvent system consisting of chloroform, methanol, water, and ammonium hydroxide was employed to produce a reliable, efficient purification of the two lipids. Lipid mass was quantified by a sensitive evaporative light-scattering detector. Using this new method, insulin addition increased both PA (87%) and LPA (217%) mass in Xenopus laevis oocytes.