Proteomics and microscopy tools for the study of antimicrobial resistance and germination mechanisms of bacterial spores.

Bacterial spores are ubiquitous in nature and can withstand both chemical and physical stresses. Spores can survive food preservation processes and upon outgrowth cause food spoilage as well as safety risks. The heterogeneous germination and outgrowth behavior of isogenic spore populations exacerbates this risk. A major unknown factor of spores is likely to be the inherently heterogeneous spore protein composition. The proteomics methods discussed here help in broadening the knowledge about spore structure and identification of putative target proteins from spores of different spore formers. Approaches to synchronize Bacillus subtilis spore formation, and to analyze spore proteins as well as the physiology of spore germination and outgrowth are also discussed. Live-imaging and fluorescence microscopy techniques discussed here allow analysis, at single cell level, of the 'germinosome', the process of spore germination itself, spore outgrowth and the spore intracellular pH dynamics. For the latter, a recently published improved pHluorin (IpHluorin) under control of the ptsG promoter is applicable. While the data obtained from such tools offers novel insight in the mechanisms of bacterial spore awakening, it may also be used to probe candidate antimicrobial compounds for inhibitory effects on spore germination and strengthen microbial risk assessment.

The use of δ(2)H and δ(18)O isotopic analyses combined with chemometrics as a traceability tool for the geographical origin of bell peppers.

Two approaches were investigated to discriminate between bell peppers of different geographic origins. Firstly, δ(18)O fruit water and corresponding source water were analyzed and correlated to the regional GNIP (Global Network of Isotopes in Precipitation) values. The water and GNIP data showed good correlation with the pepper data, with constant isotope fractionation of about -4. Secondly, compound-specific stable hydrogen isotope data was used for classification. Using n-alkane fingerprinting data, both linear discriminant analysis (LDA) and a likelihood-based classification, using the kernel-density smoothed data, were developed to discriminate between peppers from different origins. Both methods were evaluated using the δ(2)H values and n-alkanes relative composition as variables. Misclassification rates were calculated using a Monte-Carlo 5-fold cross-validation procedure. Comparable overall classification performance was achieved, however, the two methods showed sensitivity to different samples. The combined values of δ(2)H IRMS, and complimentary information regarding the relative abundance of four main alkanes in bell pepper fruit water, has proven effective for geographic origin discrimination. Evaluation of the rarity of observing particular ranges for these characteristics could be used to make quantitative assertions regarding geographic origin of bell peppers and, therefore, have a role in verifying compliance with labeling of geographical origin.

Determination of n-alkanes in C. annuum (bell pepper) fruit and seed using GC-MS: comparison of extraction methods and application to samples of different geographical origin.

An efficient extraction and analysis method was developed for the isolation and quantification of n-alkanes from bell peppers of different geographical locations. Five extraction techniques, i.e., accelerated solvent extraction (ASE), ball mill extraction, ultrasonication, rinsing, and shaking, were quantitatively compared using gas chromatography coupled to mass spectrometry (GC-MS). Rinsing of the surface wax layer of freeze-dried bell peppers with chloroform proved to be a relatively quick and easy method to efficiently extract the main n-alkanes C27, C29, C31, and C33. A combined cleanup and fractionation approach on Teflon-coated silica SPE columns resulted in clean chromatograms and gave reproducible results (recoveries 90-95 %). The GC-MS method was reproducible (R(2) = 0.994-0.997, peak area standard deviation = 2-5%) and sensitive (LODs, S/N = 3, 0.05-0.15 ng/µL). The total main n-alkane concentrations were in the range of 5-50 µg/g dry weight. Seed extractions resulted in much lower total amounts of extracted n-alkanes compared to flesh and surface extractions, demonstrating the need for further improvement of pre-concentration and cleanup. The method was applied to 131 pepper samples from four different countries, and by using the relative n-alkane concentration ratios, Dutch peppers could be discriminated from those of the other countries, with the exception of peppers from the same cultivar. Graphical Abstract Procedure for pepper origin determination.

Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms.

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Changes in the redox state and composition of the quinone pool of Escherichia coli during aerobic batch-culture growth.

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

Characterisation of UV-cured acrylate networks by means of hydrolysis followed by aqueous size-exclusion combined with reversed-phase chromatography.

UV-cured networks prepared from mixtures of di-functional (polyethylene-glycol di-acrylate) and mono-functional (2-ethylhexyl acrylate) acrylates were analysed after hydrolysis, by aqueous size-exclusion chromatography coupled to on-line reversed-phase liquid-chromatography. The mean network density and the fraction of dangling chain ends of these networks were varied by changing the concentration of mono-functional acrylate. The amount and the molar-mass distribution of the polyethylene-glycol chains between cross-links (M(XL)) and polyacrylic acid (PAA) backbone chains (the so-called kinetic chain length (kcl)) in the different acrylate networks were determined quantitatively. The molar-mass distribution of kcl revealed an almost linear dependence on the concentration of mono-functional acrylate. Analysis of the starting materials showed a significant concentration of mono-functional polyethylene-glycol acrylate. In combination with the analysis of the extractables of the UV-cured networks (polymers not attached to the network, impurities that originate from the photo-initiator and unreacted monomers), more insight in the total network structure was obtained. It was shown that the UV-cured networks contain only small fractions of residual compounds. With these results, the chemical network structure for the different UV-cured acrylate polymers was expressed in network parameters such as the number of PAA units which are cross-linked, the degree of cross-linking, and the network density, which is the molar concentration of effective network chains between cross-links per volume of the polymers. The mean molar mass of chains between chemical network junctions (M(C)) was calculated and compared with results obtained from solid-state NMR and DMA. The mean molar mass of chains between network junctions as determined by these methods was similar.

In-source decay of hyperbranched polyesteramides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hyperbranched polyesteramides (DA2), prepared from hexahydrophthalic anhydride (D) and diisopropanolamine (A) have been characterized, by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), field desorption (FD)-MS, and electrospray ionization (ESI)-MS. MALDI of polyesteramides produces protonated molecules. The spectra show a complex chemical composition distribution and end-group distribution which are mainly composed of two series of homologous oligomers DnA(n)+1 - mH2O and DnA(n) - mH2O, where m = 1-2. Signals from protonated molecules DnAn+1 and DnAn are almost absent in the MALDI spectrum, whereas these ions are responsible for the base peak of DnA(n)+1 - mH2O and DnA(n) - mH2O (m = 1-2) clusters in the ESI spectrum. The absence of -OH end-groups signals in the MALDI spectrum is due to a metastable decay of protonated DnA(n)+1 and DnAn ions in the ion source of the MALDI mass spectrometer prior to ion extraction. In-source decay results in the formation of protonated lower DnA(n)+1 - mH2O and DnA(n) - mH2O oligomers and their corresponding neutrals, leading to wrong conclusions concerning the relative end-group distribution as a function of the degree of polymerization and the chemical composition.

Determination of Block Length Distributions of Poly(oxypropylene) and Poly(oxyethylene) Block Copolymers by MALDI-FTICR Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze the block length distributions of triblock polymers of poly(oxypropylene) and poly(oxyethylene). The first series of results presented demonstrate that the apparent molecular weight distributions are distorted. This distortion is induced by the flight-time-induced mass discrimination inherent in the experimental technique, the variation of isotopic patterns over the measured mass range, and the overlap of peaks in the spectrum. Subsequently, a method for the treatment of molecular weight distributions measured by MALDI on an external ion source FTICR-MS is developed to yield the actual molecular weight distribution and, from that, the individual block length distributions. For the first time, detailed and accurate molecular weight data were obtained on a complex sample using this methodology, which independently validates the data provided by the manufacturer. The experimentally verified random coupling hypothesis proves the validity of the methodology.

Comparing mass spectrometric characteristics of peptides and peptoids.

The collision-induced dissociation (CID) spectra of the [M+H]+ ions of a pentapeptide and the corresponding peptoid and retropeptoid have been compared. The spectra of the peptide and peptoid both exhibit B- and Y"-type sequence ions at identical m/z values. In contrast to the peptide, the [M+H]+ ion of the peptoid and all sequence ions containing an N-substituted glycine derivative corresponding to a tyrosine amino acid residue can easily lose a C7H6O molecule in a charge-remote fragmentation process. The presence of N-substituted glycine residues in a peptoid is further apparent from the presence of N-substituted immonium ions, which differ significantly in their fragmentation behaviour from the corresponding immonium ions observed in the spectra of common oligopeptides. Loss of the CH2 = NH imine molecule is the dominant fragmentation reaction in the CID spectra of all peptoid immonium ions investigated in this study. The elimination of the CH = NH2 ylide analogue from common peptide immonium ions is energetically less favourable as shown by ab initio calculations. The relative heat of formation of the CH = NH2 ylide neutral appeared to be 168 kJ mol-1 more than that of the CH2 = NH imine molecule.

High resolution end group determination of low molecular weight polymers by matrix-assisted laser desorption ionization on an external ion source fourier transform ion cyclotron resonance mass spectrometer.

Matrix-assisted laser desorption ionization was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7-T superconducting magnet to analyze end groups of synthetic polymers in the mass range from 500 to 5000 u. Native, perdeutero methylated, propylated, and acetylated polyethylene glycol and polyvinyl pyrrolidone with unknown end-group elemental composition were investigated in the mass range up to 5000 u by using a 2,5-dihydroxybenzoic acid matrix. A small electrospray setup was used for the deposition of the samples. Two methods to process data were evaluated for the determination of end groups from the measured masses of the component molecules in the molecular weight ranges: a regression method and an averaging method. The averaging method is demonstrated to allow end-group mass determinations with an accuracy within 3 mu for the molecular weight range from 500 to 1400 and within 20 mu for the molecular weight range from 3400 to 5000. This is sufficient to identify the elemental composition of end groups in unknown polymer samples.

Tandem mass spectrometry and nuclear magnetic resonance spectroscopy studies of Candida bombicola sophorolipids and product formed on hydrolysis by cutinase.

Natural mixtures of sophorolipids produced by the yeast Candida bombicola have been analyzed by fast atom bombardment (FAB)-MS and collision-induced dissociation (CID)-MS. Some pure components have been analysed by two-dimensional NMR spectroscopy. The presence of acidic, lactonic, and O-acetylated forms and the position of double bonds in the fatty acid part of these glycolipids can be easily inferred from positive and negative ion FAB-mass spectra. Details about position of O-acetylation can be obtained from CID mass spectra of [M+H]+ and [M-H]- ions and from the NMR spectra. Differences in CID fragmentation between protonated and sodiated molecular ions are discussed in detail. Enzymatic hydrolysis of 6',6"-di-O-acetyl sophorolipid lactone by cutinase from Fusarium solani results specifically in the removal of the 6'-O-acetyl group, whereas the 6"-O-acetyl and lactone group are resistant. This specificity is explained from a three-dimensional model of the sophorolipid generated on the basis of the short 1H,1H distances as inferred from the NMR (ROESY) spectra.

Endgroup analysis of polyethylene glycol polymers by matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry.

Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of < 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds.

High-performance thin-layer chromatography/fast atom bombardment (tandem) mass spectrometry of Pseudomonas rhamnolipids.

Several rhamnolipid preparations from Pseudomonas strains were studied by thin-layer chromatography/fast atom bombardment (TLC/FAB) mass spectrometry and TLC/FAB tandem mass spectrometry (MS/MS). The preparations were separated with normal-phase (Silica 60) and reversed-phase (RP-8) chromatography. Silica 60 plates appeared to be very useful in the separation of rhamnolipids according to the number of monosaccharide residues present. Spectra which show characteristic fragment ions could be obtained from components of mixtures with a total sample size of less than 200 ng. Chromatography on RP-8 plates gave a good separation of the rhamnolipids based on the length of the fatty acid alkyl chain. MS/MS of the sodium cationized molecules gave information about the sequence of the building blocks. Particularly, heterogeneity in beta-hydroxy fatty acid composition was determined for the principal as well as minor components present in natural rhamnolipid mixtures.

Rapid analysis of enzymatic digests of a bacterial protease of the subtilisin type and a "bio-engineered" variant by high-performance liquid chromatography-frit fast atom bombardment mass spectrometry.

Amino acid sequencing of a subtilisin-type bacterial protease and a bio-engineered variant was carried out by investigating various enzymatic digests using HPLC-frit fast atom bombardment MS methods. The fast atom bombardment mass spectral data allowed rapid identification of the enzymatically generated peptides and differentiation between both proteins. The feasibility of determining the positions and nature of mutations in the amino acid sequence depends mainly on the size of the peptides containing the modifications.

Structure identification of natural rhamnolipid mixtures by fast atom bombardment tandem mass spectrometry.

Two rhamnobiose-lipid preparations have been studied by fast atom bombardment (FAB) tandem mass spectrometry. The principal rhamnobiose-lipids contain the beta-hydroxydecanoyl-beta-hydroxydecanoate Rha-Rha-C10-C10 and the beta-hydroxytetradecanoyl-beta-hydroxytetradecanoate Rha-Rha-C14-C14. Both preparations contain minor components which are heterogenous in beta-hydroxy fatty acid composition. FAB ionization of rhamnobiose-lipids in the presence of Na+ shows the formation of both [M + Na]+, [M +2Na-H]+, [M + 3Na-2H]+ and [M - H]- ions. Tandem mass spectrometry of the [M + 2Na-H]+ and [M - H]- ions give information about the sequence of the building blocks. Particularly, heterogeneity in beta-hydroxy fatty acid composition is determined for the principal components and all the minor components present in the preparations.

Fast atom bombardment mass spectrometry of sucrose monocaprate and sucrose monolaurate.

Fast Atom Bombardment (FAB) ionization of sucrose monocaprate and sucrose monolaurate in the presence of Na+ ions shows the formation of both [M+Na]+ and [M - H]- ions. The [M+Na]+ ions undergo charge-remote fragmentations when collisionally activated at high translational energy. These charge-remote fragmentations are useful for the structural determination of the acyclic part of the glycolipid. In the negative ion mode both sucrose esters yield anions which can be attributed to the saccharide and lipid components of these glycolipids. Structural characterization of the fatty acid can be achieved as the released carboxylate anions undergo charge-remote fragmentations that are consistent with the ion chemistry of [M - H]- anions from FAB-ionized free fatty acids.

Sequence-ion studies in peptides: the generation of C" ions.

The mechanism for the formation of C"-type ions from protonated peptides, produced under conditions of fast-atom bombardment and collisional activation was investigated. Comparison of the tandem mass spectra of the [M + H]+ ions of a model peptide and the corresponding [Md + D]+ ions, in which all exchangeable hydrogens are replaced with deuterium, revealed that neither the carboxylic hydrogen nor a hydrogen from a nitrogen atom is involved in the process of migration of a hydrogen which leads to the formation of C"n-type ions. The most feasible position from which the transferred hydrogen originates is that at the first C-atom in the side-chain of the adjacent amino acid.