Rapid analysis of intact phospholipids from whole bacterial cells by matrix-assisted laser desorption/ionization mass spectrometry combined with on-probe sample pretreatment

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), utilizing an on-probe sample pretreatment, was applied to the rapid and direct detection of intact phospholipids from whole bacterial cells. The sample preparation procedure involved depositing growing bacterial colonies from culture dishes directly onto the MALDI probe followed by treatment of the sample spot with a 3 micro L aliquot of an aqueous 0.05 M solution of sodium iodide prior to the addition of a 2,5-dihydroxybenzoic acid (DHB) matrix solution (ca. 8 mg dissolved in 70% acetonitrile/30% H(2)O containing 0.1% of trifluoroacetic acid). The MALDI spectra obtained from whole bacteria cells showed a series of ions generated from bacterial phospholipids, such as phosphatidylethanol-amines (PEs) and phosphatidylglycerols (PGs), which were clearly observed as well-resolved peaks. The ranges of the observed total carbon numbers in two acyl groups for PEs and PGs (30-36 and 33-36, respectively) were in good agreement with those reported previously. Furthermore, the distinct discrimination of four species of the Enterobacteriaceae family cultured identically was achieved by using principal components analysis (PCA) conducted on the relative peak intensities of phospholipids observed from the MALDI spectra.     

Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation

Reaction efficiencies of two organic alkalis, tetramethylammonium hydroxide (TMAH) and trimethylsulfonium hydroxide (TMSH), with lipids during thermally assisted hydrolysis and methylation (THM) were examined focusing on (1) the types of lipids and (2) degree of unsaturation of fatty acid moieties. Different types of lipids such as triglycerides, phospholipids, free fatty acids and cholesteryl esters containing saturated, monounsaturated or polyunsaturated fatty acid (PUFA) residues were subjected to THM-gas chromatography (GC) in the presence of TMAH or TMSH. The obtained results revealed that the THM reaction using TMAH allowed almost quantitative methylation of saturated and monounsaturated fatty acid components independently of the classes of lipids. However, strong alkalinity of TMAH brought about isomerization and/or degradation of PUFA components. In contrast, the use of TMSH was effective to highly sensitive detection of PUFA as well as saturated and monounsaturated fatty acid components contained in triglycerides, phospholipids (phosphatidylcholines) and free fatty acids. On the other hand, TMSH was proved to react hardly with any kind of fatty acid residues in cholesteryl esters due to their steric hindrance.     

Lipid profiling of mammalian cells with in situ matrix-assisted laser desorption ionization-mass spectrometry

The analysis of cellular lipids and phospholipids has been of continuously increasing research interest due to the importance of these molecules in psychological process. In this work, a mass spectrometry-based method for direct, in situ analysis of lipids in cells was reported. Mammalian cells were directly cultured on ITO-coated glass and then analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The matrix application process was achieved by electrospray coating, which produced a homogenous layer of matrix crystal on the sample. The detection results and reproducibility are satisfactory. With this method, a profile of abundant membrane lipids is generated, which is characteristic of cell type. In conclusion, this in situ MALDI-MS cellular lipid analysis method provides a platform for sensitive and robust molecular profiling of mammalian cells.     

Lipid profiling using catalytic pyrolysis/metal oxide laser ionization-mass spectrometry

A procedure for lipid analysis using catalytic pyrolysis metal oxide laser ionization mass spectrometry (CP-MOLI MS) is described. When surface activated CaO is mixed with a lipid sample and analyzed using CP-MOLI MS, cleavage of lipids occurs by a reaction that resembles thermal hydrolysis methylation. CP-MOLI MS of monoacylglycerides (MAG), diacylglycerides (DAG), triacylglycerides (TAG), varied fatty acids on DAGs and TAGs, phospholipids, algae, and bacteria produced Ca adducts of fatty acid constituents from the respective molecular species without matrix background interference. CP-MOLI MS offers increased speed and a streamlined analysis in which intact lipids as well as their representative fatty acid constituents can be profiled on the same instrument.     

Probing proteins on functionalized silicon surfaces using matrix-assisted laser desorption/ionization mass spectrometry

Flat H-terminated Si(111) substrates modified with alkyl monolayers terminated with hydrophobic and hydrophilic functional groups were prepared using known surface functionalization methods and characterized by FTIR, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The surfaces were then used for the study of non-specific binding of proteins from complex mixtures (using standard mixture of proteins with average molecular weight approximately 6-66 kDa) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protein adsorption on these surfaces (following on-probe fractionation of the mixture) was found to be dependent on the nature of surface functional groups, and nature and pH of rinsing solutions used. The results obtained in this work demonstrate that simple silicon-based surface modifications can be effective for direct analysis of complex mixtures by MALDI-MS. Preliminary results obtained using similarly functionalized porous silicon substrates proved that such substrates are (due to their increased surface areas) better performing than flat silicon.     

Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 x 10(6) cells/mL. In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of approximately 5.0 x 10(4) cells/mL.     

Mapping and sequencing of cardiolipins from Geobacillus stearothermophilus NRS 2004/3a by positive and negative ion nanoESI-QTOF-MS and MS/MS

In the course of systematic studies on surface layer (S-layer) glycoproteins of bacilli, the chloroform/methanol extract from whole cells of Geobacillus stearothermophilus NRS 2004/3a has been submitted to MS analysis. Glucosylated cardiolipins were found as minor components of the total lipid and phospholipid mixture by de novo identification. After purification of the crude extract using a combined column chromatography/2D TLC protocol, structural investigations of components in the lipid fraction by high resolution ESI-QTOF MS analysis provided evidence about homologous molecules attributable to the cardiolipin species containing a glycosylated backbone, and about a diversity of ester-linked fatty acid substituents. In comparative studies by positive and negative ion nanoESI-QTOF-CID-MS, maps of cardiolipin molecular ions were obtained, followed by MS/MS of the most abundant species, to provide structural details of D-glucopyranosylcardiolipin and the fatty acid substituent patterns. Experiments of the parent ion scan type revealed the presence of fatty acid moieties as isobaric combinations, represented in single molecular ion species.     

Lipids ofViburnum opulus seeds

Lipid components of the seeds ofViburnum opulus (Caprifoliaceae family) were investigated. The neutral lipids consist of eight classes, the glycolipids consist of three classes, and the phospholipids contain seven classes. The fatty-acid contents of all of the acyl-containing lipids were determined. The 18∶2 fatty acid is the main component of all the lipid fractions. The content of saturated acids is greater in the glycolipids and phospholipids. The lipophilic components, higher fatty alcohols and sterols, were identified.     

Lipids and flavolignans from <Emphasis Type="Italic">Silybum marianum</Emphasis>

Lipids from the air-dried aerial part (AP) and seeds of Silybum marianum (L.) Gaerth. (Asteraceae) were studied. The class and fatty acid compositions of neutral lipids (AP, seeds) and glyco- and phospholipids (AP) were determined. Neutral lipids (NL) with a complicated set of lipophilic components, mainly triterpenols, sterols, and their esters predominated in the AP. The fatty acids of the AP were dominated by 16:0, 18:2 (glycolipids), and 18:3 (neutral lipids, phospholipids); of seed NL, by 18:2 and 18:1. The content and composition of flavolignans isolated from defatted seeds and the content of total protein in the meal were found.     

Molecular weight determination of hyaluronic acid by gel filtration chromatography coupled to matrix-assisted laser desorption ionization mass spectrometry.

An analytical approach has been described for the molecular weight characterization of enzymatically degraded hyaluronic acid (HA). The approach involved the combined use of aqueous gel filtration chromatography (GFC) with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Microfractions were collected across an eluting peak from the chromatography system, followed by mass spectrometric analysis of these narrow fractions. The molecular mass determined by MALDI-MS and the signal obtained from the chromatography established a calibration curve for other hyaluronic acid samples analyzed by this GFC system. Results of one HA sample were obtained from both the calibration curve and direct fraction-by-fraction MALDI-MS analysis, and comparison of these results showed reasonable agreement. In contrast, molecular weights resulted from external calibration using dextran and pullullan standards showed drastically different numbers. Therefore, the GFC-MALDI-MS approach is a reliable method for the molecular weight characterization of polydisperse polysaccharides for which suitable calibration standards are unavailable for conventional GFC analysis.     

Direct fatty acid profiling of complex lipids in intact algae by fast-atom-bombardment mass spectrometry

Fast-atom-bombardment mass spectrometry (FABMS) is used for the semiquantitative determination of the fatty acids of complex lipids directly from intact algal cells, crude algal lipid extracts, and vegetable oils. Carboxylate ions, RCOO^?, corresponding to the fatty acid moieties of the complex lipids are detected. The relative abundances of the carboxylate fatty acid ions in the FAB mass spectra agreed with the relative percentages found by gas chromatography of the fatty acid methyl esters derived from the extracted lipids. Chemical ionization/fast-atom-bombardment mass spectrometry (CI/FABMS) is discussed with respect to enhancing the molecular ions of the fatty acids and triacylglycerols from these materials. The use of FABMS requires little sample preparation, and FABMS enables rapid fatty acid screening, directly from crude biological materials.     

Imaging of Phospholipids in Formalin Fixed Rat Brain Sections by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.     

Antioxidant properties of natural substances in irradiated fresh poultry

This study was undertaken to determine if a combined treatment (marinating in natural plant extracts or vacuum) with irradiation could have a synergistic effect, in order to prevent the lipid oxidation resulting in the development of undesirable flavours. The fresh chicken legs were irradiated at 0,3 and 5 kGy. The fatty acids composition of lipids was identified using gas liquid chromatography. The effect of irradiation treatment combined with a pre-treatment on the fatty acids composition was followed. The day after irradiation, ten panallists were asked to evaluate, using the instruction scaling, the overall appearance, the odor, the flavor and the overall acceptability of the samples. The major fatty acids identified in lipids were oleic acid, palmitic acid, palmitoleic acid and stearic acid. Pre-treatments have a significant effect on linoleic acid (C18:2) and higher fatty acids. The unsaturated fatty acids derived from phospholipids appeared to be more affected by the irradiation dose: however, marinating have better protection on C18:2 derived from phospholipids. The results of sensory evaluation have shown a significant better odor and flavor for the irradiated chicken at 5 kGy than the control. No significant difference have been found between the marinated chicken, the chicken irradiated under vacuum and the control.     

Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS

Ions attributed to lipids and phospholipids are directly observed by desorption from whole bacteria using intact cell (IC) matrix-assisted laser desorption-ionization (MALDI) Fourier transform mass spectrometry (FTMS). Saccharomyces cerevisiae are grown in rich media broth, concentrated, and applied directly to the MALDI surface without lysis or chemical treatment. FTMS of MALDI ions gives excellent signal to noise ratios with typical resolving powers of 90,000 and mass precision better than 0.002 Da. Use of accurate mass measurements and a simple set of rules allow assignment of major peaks into one of twelve expected lipid classes. Subsequently, fractional mass versus whole number mass plots are employed to enhance visual interpretation of the high-resolution data and to facilitate detection of related ions such as those representing homologous series or different degrees of unsaturation. This approach, coupled with rules based on bacterial biochemistry, is used to classify ions with m/z up to about 1000. Major spectral peaks in the range m/z 200-1000 are assigned as lipids and phospholipids. In this study, it is assumed that biologically-derived ions with m/z values lower than 1000 are lipids. This is not unreasonable in view of the facts that molecular weights of lipids are almost always less than 1000 Da, that the copy numbers for lipids in a cell are higher than those for any single protein or other component, and that lipids are generally collections of distinct homologous partners, unlike proteins or other cell components. This paper presents a new rapid lipid-profiling method based on IC MALDI-FTMS.     

The effects of electron and chemical Ionization Modes on the MS Profiling of Whole Bacteria

Free fatty acid profiling of whole bacteria [Francisella tularensis, Brucella melitensis, Yersinia pestis, Bacillus anthracis (vegetative and sporulated), and Bacillus cereus] was carried out with direct probe mass spectrometry under 70-eV electron ionization (EI) and isobutane chemical ionization in both the positive (CI+) and negative modes (CI-). Electron ionization produced spectra that contained molecular ions and fragment ions from various free fatty acids. Spectra acquired with isobutane chemical ionization in the positive mode yielded molecular ions of free fatty acids as well as ions from other bacterial compounds not observed under EI conditions. Spectra obtained with negative chemical ionization did not contain as much taxonomic information as EI or CI+; however, some taxonomically significant compounds such as dipicolinic acid and poly(3-hydroxybutyrate) did produce negative ions. All ionization modes yielded spectra that could separate the bacteria by Gram-type when observed with principle components analysis (PCA). Chemical ionization in the positive ion mode produced the greatest amount of differentiation between the four genera of bacteria when the spectra where examined by PCA.     

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was ∼90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were <0.002-0.29% of total lipids from camembert, <0.002-0.12% of total lipids from mozzarella, and <0.002-0.18% of total lipids in a goat cream cheese. Differences in the fatty acid pattern of neutral and polar lipids were detected. The quantity of the fatty acids determined in the phospholipid fraction was divided by the factor 0.7 in order to convert the fatty acid content into the phospholipid content of the cheese samples. This factor is based on the contribution of 16:0 to dipalmitoylphosphatidylcholine (DPPC). The resulting DPPC equivalents (DPPC_eq) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese was 0.60%, 1.42% and 0.79%, respectively.     

Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20 kDa) proteins

Three sample preparation strategies commonly employed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS) of whole bacterial cells were investigated for the detection of high mass signals; these included the dried droplet, the seed-layer/two-layer, and the bottom-layer methods. Different sample preparation approaches favoured the detection of high- or low-mass proteins. The low-mass peaks were best detected using the bottom-layer method. By contrast, the dried droplet method using a solvent with higher water content, and hence effecting a slower crystallization process, gave the best results for the detection of high-mass signals. Signals up to m/z 158 000 could be detected with this methodology for Bacillus sphaericus. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the same extracts used for MALDI-TOFMS showed bands in the molecular weight range in which high-mass peaks were observed in MALDI-MS, suggesting that the high-mass signals are not polymeric adducts of low-mass protein monomers. In addition, one of the high molecular weight proteins (approximately 126 kDa) was putatively identified as an S-layer protein by an in-gel tryptic digest. The bacterial samples spotted on the target wells for MALDI-TOFMS, using the different sample preparation strategies, were examined under a scanning electron microscope and differences were observed between the different strategies, suggesting that the nature of the crystals and the distribution of the analytes amidst the crystals could influence the spectral pattern observed in MALDI-TOFMS of whole bacterial cells. Finally, evidence is presented to indicate that, although the determinants are intact cells, cell lysis occurs both before and during the MALDI process.     

In situ structural characterization of glycerophospholipids and sulfatides in brain tissue using MALDI-MS/MS

Lipids are major structural components of biomembranes. Negatively charged species such as phosphatidylinositol, phosphatidylserine, sulfatides, and the zwitterionic phosphatidylethanolamines are major components of the cytoplasmic surface of the cellular membrane lipid bilayer and play a key role in several receptors signaling functions. Lipids are not just involved in metabolic and neurological diseases; negatively charged lipids in particular play crucial roles in physiological events such as signal transduction, receptors, and enzymatic activation, as well as storage and release of therapeutic drugs and toxic chemicals in the body. Due to the importance of their role in signaling, the field of lipidomics has rapidly expanded in recent years. In the present study, direct probing of tissue slices with negative ion mode matrix assisted laser desorption/ionization mass spectrometry was employed to profile the distribution of lipids in the brain. In total, 32 lipid species consisting of phosphatidylethanolamines, phosphatidylglycerol, phosphatidylinositols, phosphatidylserines, and sulfatides were assigned. To confirm the structure of lipid species, MALDI-MS/MS analysis was conducted. Product-ion spectra obtained in negative ion mode allow for the assignment of the head groups and the fatty acid chains for the lipid species.     

Determination of the acyl moieties of the antibiotic complex A40926 and their relation with the membrane lipids of the producer strain.

Structures of the fatty acid residues characterizing the various components of A40926 were determined by gas chromatography/mass spectrometry on the methyl esters obtained by methanolysis of the complex. The results confirm the residues previously assigned to Factor A (n-undecanoic acid) and B (10-methyl-undecanoic acid) and establish the residues of Factor A1 (9-methyl-decanoic acid), B1 (n-dodecanoic acid), RS1 (8-methyl-nonanoic acid), RS2 (n-decanoic acid), and RS3 (n-tridecanoic acid). As the Actinomadura species contain in their mycelia large quantities of C15-C17 fatty acid residues as membrane phospholipids, these mycelia were saponified and the fatty acids obtained were analyzed as above. There is a close correlation between the fatty acid content of A40926 complex and that of the longer homologues in the producer mycelia.     

Integration of microfiltration and anion-exchange nanoparticles-based magnetic separation with MALDI mass spectrometry for bacterial analysis

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is powerful in characterizing and identifying bacterial isolates. However, sufficient quantities of bacterial cells are required for generating MALDI mass spectra and a procedure to isolate and enrich target bacteria from sample matrix prior to MALDI-MS analysis is often necessary. In this paper, anion-exchange superparamagnetic nanoparticles (NPs), i.e., fluidMAG-DEAE and fluidMAG-Q, were employed to capture Aeromonas, Salmonella, Pseudomonas, Enterococcus, Bacillus, Staphylococcus and Escherichia coli from aqueous solutions and fresh water. The magnetically isolated bacteria were then characterized by whole cell MALDI-MS. The capture efficiency was found to be dependent on bacterial species, medium pH, the functional group and concentration of the NPs. The experimental results demonstrated that fluidMAG-DEAE and fluidMAG-Q were broad spectrum probes for bacteria. Furthermore, both dead and live bacteria could be captured by the NPs, and the live bacteria captured remained viable. Membrane filtration prior to the magnetic isolation could increase enrichment factor and eliminate potential matrix interference. A detection limit of 1 × 10³ cfu/ml was achieved for the bacteria spiked in tap water and reservoir water, and the analytical time was around 2 h.