Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.     

Analysis of polyunsaturated aminophospholipid molecular species using isotope-tagged derivatives and tandem mass spectrometry/mass spectrometry/mass spectrometry

When aminophospholipids with only saturated and monounsaturated fatty acids esterified to the glycerol backbone were labeled with isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, it was found that they could be readily detected as N-methylpiperazine-amide-tagged aminophospholipids using a precursor scan of the stable isotope reporter ion (m/z 114-117) formed by tandem mass spectrometry/mass spectrometry. However, it was found in the current study that these precursor ion scans are not useful in determining the changes of aminophospholipids with polyunsaturated fatty acids (PUFAs) esterified to the glycerol backbone due to the presence of interfering ions in the reporter ion region. Therefore, a method was developed using tandem mass spectrometry/mass spectrometry/mass spectrometry (MS(3)) to obtain reporter ion ratios that were not distorted by interfering ions present in the collision-induced dissociation spectra of nontagged aminophospholipids with PUFAs. This new MS(3) method for N-methylpiperazine- amide-tagged aminophospholipids was used to examine the fate of diacyl, ether, or plasmalogen glycerophosphoethanolamine (GPEtn) species after exposure of human polymorphonuclear leukocytes to A23187 and granulocyte macrophage-colony-stimulating factor/formyl-methionyl-leucyl-phenylalanine stimuli, which can induce eicosanoid biosynthesis, to follow those GPEtn molecular species which were the source of arachidonic acid released. Upon stimulation of the human polymorphonuclear leukocyte, it was found that the abundant arachidonoyl GPEtn plasmalogen molecular species were uniquely reduced in relative content compared to ether or diacyl species and this subclass of GPEtn may be a source of the arachidonic acid converted to leukotrienes by the 5-lipoxygenase pathway activated in this cell.     

Imaging mass spectrometry detection of gangliosides species in the mouse brain following transient focal cerebral ischemia and long-term recovery

Gangliosides, a member of the glycosphingolipid family, are heterogeneously expressed in biological membranes and are particularly enriched within the central nervous system. Gangliosides consist of mono- or poly-sialylated oligosaccharide chains of variable lengths attached to a ceramide unit and are found to be intimately involved in brain disease development. The purpose of this study is to examine the spatial profile of ganglioside species using matrix-assisted laser desorption/ionization (MALDI) imaging (IMS) following middle cerebral artery occlusion (MCAO) reperfusion injury in the mouse. IMS is a powerful method to not only discriminate gangliosides by their oligosaccharide components, but also by their carbon length within their sphingosine base. Mice were subjected to a 30 min unilateral MCAO followed by long-term survival (up to 28 days of reperfusion). Brain sections were sprayed with the matrix 5-Chloro-2-mercaptobenzothiazole, scanned and analyzed for a series of ganglioside molecules using an Applied Biosystems 4800 MALDI TOF/TOF. Traditional histological and immunofluorescence techniques were performed to assess brain tissue damage and verification of the expression of gangliosides of interest. Results revealed a unique anatomical profile of GM1, GD1 and GT1b (d18:1, d20:1 as well as other members of the glycosphingolipid family). There was marked variability in the ratio of expression between ipsilateral and contralateral cortices for the various detected ganglioside species following MCAO-reperfusion injury. Most interestingly, MCAO resulted in the transient induction of both GM2 and GM3 signals within the ipsilateral hemisphere; at the border of the infarcted tissue. Taken together, the data suggest that brain region specific expression of gangliosides, particularly with respect to hydrocarbon length, may play a role in neuronal responses to injury.     

Application of liquid chromatography—thermospray mass spectrometry in the analysis of glycerophospholipid molecular species

We report the application of high-performance liquid chromatographic (HPLC) separation with ultraviolet detection and direct, on-line, structural analyses by mass spectrometry of glycerobenzoate derivatives from complex mixtures of phospholipid molecular species. Individual phospholipids were resolved from total lipid extracts by thin-layer chromatography (TLC). Diradylglycerols were released from phospholipids by phospholipase-C treatment, converted to diradyl glycerobenzoates and subsequently separated by TLC into subclasses (alk-1-enylacyl, alkylacyl and diacyl types). The molecular species within each subclass were resolved by HPLC with an octadecyl reversed-phase column in acetonitrile—isopropanol (80:20, v/v). Individual peaks were quantitated at the picomole level by measuring absorbance at 230 nm. After post-column addition of methanol—0.2 M ammonium acetate (50:50, v/v), peaks were introduced through the thermospray interface into a VG Masslab 30–250 quadrupole mass spectrometer. Molecular species showed as base peaks the salt adducts of the molecular ion which permitted easy deduction of the overall fatty acyl composition. In addition, the diglyceride fragment of each species was found at [MH — 122]⁺ and two fragments formed by the loss of the fatty acyl groups (R) in the sn-1 or sn-2 position were found at [M — R₁]⁺ and [M — R₂]⁺, respectively. Since preferential release of either fatty acyl group was observed in positional isomers, the ratio of the intensity of these fragments gave information on the position of the fatty acyl groups in the individual HPLC peaks. We show that the use of on-line mass spectrometry, however, provides easy identification of all molecular species present in a complex phospholipid mixture, even when more than one molecular species is contained in an HPLC peak.     

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w') and fatty acyl chains (Y0'-w') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.     

MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data

SUMMARY: New additional methods are presented for processing and visualizing mass spectrometry based molecular profile data, implemented as part of the recently introduced MZmine software. They include new features and extensions such as support for mzXML data format, capability to perform batch processing for large number of files, support for parallel processing, new methods for calculating peak areas using post-alignment peak picking algorithm and implementation of Sammon's mapping and curvilinear distance analysis for data visualization and exploratory analysis. AVAILABILITY: MZmine is available under GNU Public license from http://mzmine.sourceforge.net/.     

Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.     

Diversity of Clonostachys species assessed by molecular phylogenetics and MALDI-TOF mass spectrometry

We assessed the species diversity among 45 strains of Clonostachys from different substrates and localities in Brazil using molecular phylogenetics, and compared the results with the phenotypic classification of strains obtained from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analyses were based on beta tubulin (Tub), ITS-LSU rDNA, and a combined Tub-ITS DNA dataset. MALDI-TOF MS analyses were performed using intact conidia and conidiophores of strains cultivated on oatmeal agar and 4 % malt extract agar. Six known species were identified: Clonostachys byssicola, Clonostachys candelabrum, Clonostachys pseudochroleuca, Clonostachys rhizophaga, Clonostachys rogersoniana, and Clonostachys rosea. Two clades and two singleton lineages did not correspond to known species represented in the reference DNA dataset and were identified as Clonostachys sp. 1–4. Multivariate cluster analyses of MALDI-TOF MS data classified the strains into eight clusters and three singletons, corresponding to the ten identified species plus one additional cluster containing two strains of C. rogersoniana that split from the other co-specific strains. The consistent results of MALDI-TOF MS supported the identification of strains assigned to C. byssicola and C. pseudochroleuca, which did not form well supported clades in all phylogenetic analyses, but formed distinct clusters in the MALDI-TOF dendrograms.     

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH(3). Deuterium-labeled internal standards were used to normalize the signal for each nominal [M+NH(4)](+) ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo(2)-lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo(2)-lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.     

Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry

Glycerophospholipids (GPL) in animal tissues are composed of a large array of molecular species that mainly differ in the fatty acyl composition. In order to further understand the roles of GPL at the molecular level, it is necessary to have comprehensive, accurate accounts of the molecular makeup for these molecules in animal tissues. However, this task was difficult simply because the conventional technologies of profiling GPL species depended heavily on technical skill for accuracy and reliability and were extremely labor-intensive. In recent years, tandem mass spectrometry (MS/MS) proved to be a highly reliable and sensitive technology for profiling small molecules, including GPL, in biological samples. In this study, we used this technology to perform simultaneous comparative analyses for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) in the same lipid preparations of liver, lung, kidney, heart, pancreas, stomach, small intestine, spleen, skeleton muscle and brain of an adult rat. We produced molecular profiles of these 4 GPL classes in these 10 different tissues that are highly reproducible between different scans of the same sample and between samples from different animals. It is intriguing that each tissue was found to possess a unique signature of GPL profile that may be used to identify unknown tissues. More importantly, these profiles may also set reference points for studying changes of GPL metabolism in different physiological and pathological conditions.     

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.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Normal phase liquid chromatography-electrospray ionization tandem mass spectrometry analysis of phospholipid molecular species in blood mononuclear cells: application to cystic fibrosis

The use of HPLC coupled on-line with a mass spectrometer is a very powerful tool in order to analyze intact PLs molecular species (PMS) without the need of derivatization, thus decreasing the risk of artifacts formation. A normal-phase HPLC-ESI-MS-MS method has been developed in order to study the human blood mononuclear cell PMS composition. This method was applied to characterize PMS from seven CF subjects and from seven age-matched healthy subjects. More than 140 phospholipid molecular species from phosphatidylethanolamine (PE), plasmalogen phosphatidylethanolamine (pPE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (Sph) were identified and compared. Differences between the two groups were found in pPE (p16:0/22:6), pPE (p18:0/22:6), PE (16:0/20:4) and PC (16:0/18:2) which were significantly lower in CF subjects and in PC (16:0/16:1) which was significantly higher in CF subjects.     

Quantitation of cardiolipin molecular species in spontaneously hypertensive heart failure rats using electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1',2,2'-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]-) current from four different CL reference standards and the [M-H]- from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples. The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased.     

Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures

Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties.     

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid-liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 x 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/z 854.4--> 286.2 transition of paclitaxel is measured versus that of the m/z 808.5--> 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2-500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (< or = 15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy < or = 15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.     

The application of hybrid mass spectrometry/mass spectrometry and high-resolution mass spectrometry to the analysis of fish samples for polychlorinated dibenzo-p-dioxins and dibenzofurans

A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility.