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.     

Invertebrate and protozoan epibionts on the velvet swimming crab Liocarcinus puber (Linnaeus, 1767) from Scotland

Invertebrate and ciliate protozoan epibionts of velvet swimming crabs collected near Millport (Scotland) were analysed. The ecdysis peak for male crabs was at the time of collection while that for female crabs was 2 months later. The epibionts were: the polychaetes Pomatoceros triqueter and Hydroides norvegica, the cirriped Balanus crenatus, the entoproct Barentsia matsushimana, the hydroids Leuckartiara sp. and Clytia sp., and the ciliate protozoans Ephelota plana, Ephelota gemmipara, Chilodochona quennerstedti and Cothurnia longipes. The polychaetes, cirripeds, entoprocts and hydroids, all of them with comparatively larger size, were distributed on the carapace, ventral surface of the cephalothorax and the pereiopods; meanwhile the protozoans, with smaller size, were attached also on pleopods, antennae, eyes and buccal appendages. Chilodochona quennerstedti was the epibiont most abundant on the crab, followed by Ephelota plana. Cothurnia longipes was, in contrast, the least abundant epibiont, followed by Barentsia matsushimana. The anatomical unit most colonized was the left third maxilliped, followed by the left first maxilliped and the ventral surface. The less colonized anatomical units were the left antenna and the ocular orbits. Epibionts on this crab have not been described before. Statistical analyses of the epibiont distribution on the crab were carried out. There were significant correlations between right and left appendages in 66.67% of the cases. Males and females differed significantly with respect to the distribution of epibionts on their anatomical units. The comparison analysis indicated a significant differential distribution of each epibiont species on the anatomical units of the crab. Principal component analysis grouped the epibiont species according to their colonization pattern in three clusters: (1) Ephelota plana, Ephelota gemmipara and Chilodochona quennerstedti; (2) Leuckartiara sp., Clytia sp., Barentsia matsushimana and Cothurnia longipes; and (3) Pomatoceros triqueter, Balanus crenatus and Hydroides norvegica. The hierarchical cluster analysis grouped the anatomical units of the crab in relation to their colonization in five clusters.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy)

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.     

Intracellular boron localization and uptake in cell cultures using imaging secondary ion mass spectrometry (ion microscopy) for neutron capture therapy for cancer.

Quantitative ion microscopy of freeze-fractured, freeze-dried cultured cells is a technique for single cell and subcellular elemental analysis. This review describes the technique and its usefulness in determining the uptake and subcellular distribution of the boron from boron neutron capture therapy drugs.     

Cellular and subcellular distribution of iron in the lamina propria of rat duodenum

Summary Cellular and subcellular distribution of iron in the lamina propria of rat duodenum was studied after a single i.p. injection of iron dextran, using electron microscopy and peroxidase cytochemistry. X-ray spectrum microanalysis was used for positive identification of iron. Ironcontaining particles (IP) were found in the cytoplasm of three cell types, viz. macrophages, pericytic reticular cells and sheathing fibrocytes. IP-containing organelles in lamina propria cells were more heterogeneous compared to absorptive cells and, in addition, some differences were noted in the subcellular distribution of IP in the 3 cell types. A common denominator in these 3 cell types was the presence of endogenous peroxidase, also shared by Kupffer cells which are known to be involved in iron storage. Peroxidase activity was absent in absorptive epithelial cells. It is hypothesized that the cells of the lamina propria, like Kupffer cells, may be the site of storage of excess iron absorbed, releasing iron upon demand and migrating into the lumen to prevent iron overload. In this fashion they may regulate the exchange of iron with the environment. The presence of peroxidase in these as well as Kupffer cells, and its absence in absorptive cells also raises the possibility that this enzyme may be related to certain aspects of iron storing process.     

Secondary ion mass spectrometry analysis of the copper distribution in Drosophila melanogaster chronically intoxicated with Bordeaux mixture

The fungicides used intensively in agriculture may affect non-target organisms. The concentrations of copper sulfate-based fungicide, Bordeaux mixture, normally used in agriculture, can significantly reduce both the life span and breeding rate of Drosophila melanogaster. The present study examines the distribution of copper in organ sections of fruit flies intoxicated with Bordeaux mixture, by secondary ion mass spectrometry. The organs of most control flies contained no copper. In contrast, copper accumulated in the cytoplasm of all the mesenteron and Malpighian tubule epithelial cells of the treated flies. There were also copper deposits in the fat body and the epithelia of the seminal receptacle and accessory glands of some flies, but there was little or no copper in the ovaries. The mesenteron and Malpighian tubules are generally responsible for detoxification by accumulation of ingested metal salts in insects. The high concentration of Bordeaux mixture used saturated these organs and resulted in excess copper being deposited in other sites, such as the fat body and the reproductive system.     

The synthesis and mass spectrometry of oligonucleotides bearing thiophosphoryl modifications of the predetermined localization

The technique of parallel automated synthesis of oligodeoxynucleotides bearing various local thiophosphoryl internucleotide bonds was optimized using assembling in the standby mode and creation of special program blocks. The selected conditions of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF MS) provided an increase in the method sensitivity (up to 1–10 fmol of oligonucleotide in sample) and registration of reliable spectra of oligodeoxynucleotide thiophosphoryl analogues. This enables to reliably prove the presence of the specified number of thiophosphoryl bonds within synthetic sequences. A series of oligodeoxynucleotides, thioanalogues of d(GGTTGGTGTGGTTGG), a known G-quadruplex antithrombin aptamer, were obtained.     

Convenient and rapid analysis of linkage isomers of fucose-containing oligosaccharides by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSⁿ spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.     

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.     

Detection of volatile compounds produced by microbial growth in urine by selected ion flow tube mass spectrometry (SIFT-MS)

Selected ion flow tube-mass spectrometry has been used to measure the volatile compounds occurring in the headspace of urine samples inoculated with common urinary tract infection (UTI)-causing microbes Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterococcus faecalis, or Candida albicans. This technique has the potential to offer rapid and simple diagnosis of the causative agent of UTIs.     

Cytochemistry and X-ray microprobe analysis of the midgut of Tomocerus minor lubbock (Insecta,Collembola) with special reference to the physiological significance of the mineral concretions

Summary Histochemical and cytochemical analyses have been made on the mineral concretions within the midgut cells of Tomocerus minor. The classical histochemical methods are not specific and precise enough and have been supplemented with cytochemical techniques on ultrathin sections. The most interesting of these was the K-pyroantimonate technique combined with glutaraldehyde-osmium fixation. This technique shows the distribution of cations such as Ca⁺⁺, K⁺, Mg⁺⁺ and Na⁺ on the concentric layers of the concretions. Chloride ions can be detected by means of the silver lactate technique. The action of calcium chelators such as E.D.T.A. shows an important distribution of calcium ions in the concretions. The spectra obtained by electron probe microanalysis from areas of fresh, dried and carbon coated midguts as well as from carbon coated semithin or ultrathin sections reveal the presence of Ca, K, Mg, S, Cl and P principally. Other elements such as aluminium, silicon and manganese have also been detected. Iron is not always present. The chemical and X-ray analytical investigations indicate that the midgut concretions are mainly built up of calcium, potassium, magnesium and sodium phosphates, perhaps associated with chlorides and carbonates. An organic matrix formed by polysaccharides seems to join the different mineral layers. These concretions may be formed within the vesicles of rough endoplasmic reticulum. The midgut cells are highly differentiated and very active in transport. Extensive basal infoldings and apical microvilli as well as lateral membranes are a site of small cationic deposits. The possible pathway of ion transport in the cell and the physiological significance of the concretions are discussed. The principal function of these concretions seems to be the maintenance of the mineral balance and to trap foreign and excess ions.

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Résumé L'analyse chimique des sphérocristaux de l'intestin moyen de Tomocerus minor a été réalisée. Les méthodes histochimiques courantes manquant souvent de spécificité et de sensibilité ont été complétées avec des méthodes cytochimiques sur coupes ultrafines. La plus intéressante a été la technique du pyroantimonate de K montrant la distribution des cations Ca⁺⁺, K⁺, Mg⁺⁺, Na⁺ sur les couches concentriques des sphérocristaux. La technique au lactate d'argent permet de déceler les ions Cl^-. L'action d'agents chélateurs du Ca tels l'E.D.TA. montre une importante distribution du calcium dans les sphérocristaux. L'analyse spectrographique d'étalements de mésentérons séches, carbonés et de coupes semi-fines ou ultrafines carbonées montre la présence de Ca, K, Mg, S, Cl, P, Na. D'autres éléments tels l'Al et le Si ont pu être détectés. Le Fe n'est pas toujours présent. Les sphérocristaux semblent formés essentiellement de phosphates de calcium, de potassium, de magnésium, de sodium associés peut-être à des chlorures ou des carbonates. Une matrice organique constituée essentiellement par des polysaccharides semble lier les différentes couches minérales. Ces sphérocristaux prennent naissance à l'intérieur des vésicules de l'ergastoplasme. Les cellules de l'intestin moyen sont très différenciées et sont le siège de nombreux transports actifs. Les replis basaux de la membrane plasmique, les microvillosités apicales, de même que les membranes latérales sont le siège de dépôts de cations. Le transport des ions dans les cellules ainsi que le rôle physiologique des sphérocristaux sont discutés. Le maintien de la balance hydrique ainsi que le piégeage d'ions étrangers ou en surplus semblent être la principale fonction des sphérocristaux.

     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.     

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins.Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins.A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins.However,all the existing methods have inherent advantages and limitations.Up to now,there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.     

High-resolution secondary ion mass spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.     

Characterization of glyoxalase I (E. coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.     

Linking environmental processes to the in situ functioning of microorganisms by high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X‐ray microscopy (STXM)

Environmental microbiology research increasingly focuses on the single microbial cell as the defining entity that drives environmental processes. The interactions of individual microbial cells with each other, the environment and with higher organisms shape microbial communities and control the functioning of whole ecosystems. A single‐cell view of microorganisms in their natural environment requires analytical tools that measure both cell function and chemical speciation at the submicrometre scale. Here we review the technical capabilities and limitations of high‐resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission (soft) X‐ray microscopy (STXM) and give examples of their applications. Whereas NanoSIMS can be combined with isotope‐labelling, thereby localizing the distribution of cellular activities (e.g. carbon/nitrogen fixation/turnover), STXM provides information on the location and chemical speciation of metabolites and products of redox reactions. We propose the combined use of both techniques and discuss the technical challenges of their joint application. Both techniques have the potential to enhance our understanding of cellular mechanisms and activities that contribute to microbially mediated processes, such as the biogeochemical cycling of elements, the transformation of contaminants and the precipitation of mineral phases.     

Use of secondary ion mass spectrometry to image44calcium uptake in the cell walls of apple fruit

Summary Calcium, an important agent in regulating cell wall autolysis during fruit ripening, interacts with pectic acid polymers to form cross-bridges that influence cell separation. In the present study, secondary ion mass spectrometry (SIMS) was used to determine whether the cell walls of apple fruit were able to take up exogenously applied⁴⁴Ca, which was infiltrated into mature fruit. SIMS, which has the ability to discriminate between isotopes, allowed localization of the exogenously applied⁴⁴Ca and the native⁴⁰Ca. The results indicated that the total amount of calcium present in the cell walls was enriched with⁴⁴Ca and that heterogeneity of⁴⁴Ca distribution occurred in the pericarp. Isotope ratio images showed microdomains in the cell wall, particularly in the middle lamella intersects that oppose the intercellular spaces. These domains may be the key areas that control cell separation. These data suggest that exogenously applied calcium may influence cell wall autolysis.Abbreviations SIMS secondary ion mass spectrometry     

Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.