Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.     

The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549T

Novel lipooligosaccharide components were isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Pseudoalteromonas issachenkonii type strain KMM 3549T. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy and MALDI mass spectrometry as the following: [carbohydrate formula see text] All sugars are d-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked. In addition, by MALDI mass spectrometry of the intact LOS, the lipid A moiety was also identified as a mixture of penta-, tetra- and triacylated species.     

A critical comparison between two classical and a kit‐based method for mitochondria isolation

Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free‐flow electrophoresis or a commercially available kit‐based method. Our analyses show that the sophisticated (and most expensive) free‐flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time‐consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations.     

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 μg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.     

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]⁺ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.     

Simple procedure for rapid isolation of functionally intact mitochondria from human and rat skeletal muscle

A simple procedure for the rapid isolation of functionally intact skeletal muscle mitochondria is described. The method involves homogenization of muscle in a medium comprising sucrose (0.25 M) containing 50,000 units of heparin/liter, followed by differential centrifugation. Mitochondria so isolated are functionally and morphologically intact.     

Differential impact of environmental stresses on the pea mitochondrial proteome

Exposure to adverse environmental conditions causes oxidative stress in many organisms, leading either to disease and debilitation or to response and tolerance. Mitochondria are a key site of oxidative stress and of cellular response and play important roles in cell survival. We analyzed the response of mitochondria in pea (Pisum sativum) plants to the common stresses associated with drought, cold, and herbicides. These treatments all altered photosynthetic and respiratory rates of pea leaves to various extents, but only herbicides significantly increased lipid peroxidation product accumulation. Mitochondria isolated from the stressed pea plants maintained their electron transport chain activity, but changes were evident in the abundance of uncoupling proteins, non-phosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties on mitochondrial proteins. These data suggest that herbicide treatment placed a severe oxidative stress on mitochondria, whereas chilling and particularly drought were milder stresses. Detailed analysis of the soluble proteome of mitochondria by gel electrophoresis and mass spectrometry revealed differential degradation of key matrix enzymes during treatments with chilling being significantly more damaging than drought. Differential induction of heat shock proteins and specific losses of other proteins illustrated the diversity of response to these stresses at the protein level. Cross-species matching was required for mass spectrometry identification of nine proteins because only a limited number of pea cDNAs have been sequenced, and the full pea genome is not available. Blue-native separation of intact respiratory chain complexes revealed little if any change in response to environmental stresses. Together these data suggest that although many of the molecular events identified by chemical stresses of mitochondria from a range of model eukaryotes are also apparent during environmental stress of plants, their extent and significance can vary substantially.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

A method for isolating intact mitochondria and nuclei from the same homogenate,and the influence of mitochondrial destruction on the properties of cell nuclei

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0-6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl(2) is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The alpha-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0-6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.     

LC-ESI/MS determination of xanthone and secoiridoid glycosides from in vitro regenerated and in vivo Swertia chirayita

A rapid analytical method has been developed to determine xanthone and secoiridoid glycoside in in vitro and in vivo Swertia chirayita extracts. Ultra performance liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS) was applied and validated for the analysis of xanthone and secoiridoid glycoside a potential active component isolated from methanolic extracts of in vitro and in vivo Swertia chirayita plantlets. Chromatographic separation was achieved on a RP-C18 column using gradient elution. Mangiferin (Xanthone), Amarogentin and Swertiamarin (Secoiridoid glycosides) were identified in both the extracts. In the LC/ESI-MS spectra, major [M + H] ⁺ and [M + Na] ⁺ ions were observed in positive ion mode and provided molecular mass information. An ultra-performance liquid-chromatography in combination with electrospray ionization tandem mass spectrometry involving metal cationisation was successfully utilized for the rapid identification of xanthone and secoiridoid glycosides. This method is suitable for the routine analysis, as well as for the separation and identification of known and novel secoiridoid glycoside and xanthone.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Many methods have been proposed to analyze glucosinolates, a class of phytochemicals whose breakdown products are thought to be responsible for an improvement in health; however, few are quantitative and many are time consuming. A selective and sensitive quantitative method for direct determination of intact glucosinolates was developed using liquid chromatography coupled to electrospray ionization tandem mass spectrometry with selected reaction monitoring detection. Detection limits for glucoiberin, sinigrin, progoitrin, glucoerucin, and glucotropaeolin were 1.75, 1.38, 1.36, 0.6, and 0.63 pmol, respectively. Intraassay precision of the method was within 10% for each compound. The method was successfully applied to quantify 10 individual glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower. The advantage of the proposed method includes analysis of individual intact glucosinolates rather than the conversion to desulfoglucosinolates, an increased selectivity through the use of mass spectrometry, and a 10-fold improvement in detection sensitivity over conventionally used HPLC techniques.     

Characterization by mass spectrometry of blood group A active glycolipids from human and dog small intestins.

Glycolipids with blood group A activity isolated from human and dog small intestine have been characterized by mass spectrometry of intact lipid in methylated and in methylated and reduced (LiAiH4) form. Without degradative studies the glycolipids were conclusively shown to be hexaglycosyleramides with phytosphingosine as the major long-chain base and hydroxypalmitic acid as the major fatty acid. The exact sugar ratio was hexose-hexosamine-deoxyhexose 3:2:1 and the sequence established as hexosamine-[deoxyhexose-]hexose-hexosamine-hexose-hexose-ceramide. Evidence is presented that mass spectrometry can differential between type ) and type 2 saccharide chains.     

Acyl Transfer from Membrane Lipids to Peptides Is a Generic Process

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH 7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3 h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.     

Mass processing--an improved technique for protein identification with mass spectrometry data.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis following tryptic digestion of polyacrylamide gel pieces is a common technique used to identify proteins. This approach is rapid, sensitive, and user friendly, and is becoming widely available to scientists in a variety of biological fields. Here we introduce a simple and effective strategy called "mass processing" where the list of masses generated from a mass spectrometer undergoes two stages of data reduction before identification. Mass processing improves the ability to identify in-gel tryptic-digested proteins by reducing the number of nonsample masses submitted to protein identification database search engines. Our results demonstrate that mass processing improves the statistical score and rank of putative protein identifications, especially with low-quantity samples, thus increasing the ability to confidently identify proteins with mass spectrometry data.     

Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.     

Surface analysis of lipids by mass spectrometry: More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the “omics” age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.     

Isolation and characterization of O-acetylated glucomannans from aspen and birch wood

O-acetylated glucomannans were isolated from aspen and birch wood employing two different procedures and thereafter subjected to carbohydrate analysis by NMR spectroscopy and MALDI mass spectrometry. In one of the isolation procedures, acetone-extracted aspen or birch wood meal was extracted with dimethyl sulfoxide and then with hot water. Fractionation of the hemicellulose-containing extracts by size-exclusion chromatography was subsequently performed. In the other procedure, fractional precipitation with ethanol was used to isolate glucomannans from lyophilized process water produced by mechanical pulping of aspen. The aspen and birch glucomannans are O-acetylated at the C-2 or C-3 position of some of the mannose residues (random distribution), with a degree of acetylation of approx 0.3. In both cases the degree of polymerization was approx 16, indicating that low-molecular mass fractions of the glucomannans in hardwood have been isolated here.