Metabolic profiling by ion mobility mass spectrometry (IMMS)

Ion Mobility Mass Spectrometry (IMMS) was evaluated as an analytical method for metabolic profiling. The specific instrument used in these studies was a direct infusion (DI)-electrospray ionization (ESI)—ambient pressure ion mobility spectrometer (APIMS) coupled to a time-of-flight mass spectrometer (TOFMS). The addition of an ion mobility spectrometer to a mass spectrometer had several advantages over direct infusion electrospray mass spectrometry alone. This tandem instrument (ESI-IMMS) added a rapid separation step with high-resolution prior to mass spectrometric analysis of metabolite mixtures without extending sample preparation time or reducing the high through put potential of direct mass spectrometry. Further, IMMS also reduced the baseline noise common with ESI-MS analyses of complex samples and enabled rapid separation of isobaric metabolites. IMMS was used to analyze the metabolome of Escherichia coli (E. coli), containing a collection of extremely diverse chemical compounds including hydrophobic lipids, inorganic ions, volatile alcohols and ketones, amino and non-amino organic acids, and hydrophilic carbohydrates. IMMS data were collected as two-dimensional spectra showing both mobility and mass of each ion detected. Using direct infusion ESI-IMMS of a non-derivatized methanol extract of an E. coli culture, more than 500 features were detected, of which over 200 intracellular metabolites were tentatively assigned as E. coli metabolites. This analytical method also allowed simultaneous separation of isomeric metabolic features.     

Improved molecular methods to characterise Serpula lacrymans and other Basidiomycetes involved in wood decay

Early detection of indoor wood-decay fungi is crucial to prevent building deterioration and thereby avoid considerable economic loss. Due to their increased sensitivity, two reliable DNA-based fingerprinting techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) and denaturing high-performance liquid chromatography (DHPLC), were used to identify Serpula lacrymans and to profile wood-rot Basidiomycetes in the built environment. Molecular fungal diversity was assessed on 74 environmental samples, collected from 2003 to 2009 from infected buildings in France. S. lacrymans, the most widespread, indoor wood-decay fungus accounted for 64% of total wood-rot Basidiomycetes. A number of other common wood-rot fungi such as Coniophora puteana, Trametes versicolor and Donkioporia expansa were identified. Other Basidiomycetes such as Phlebiopsis gigantea and Scleroderma verrucosum were detected for the first time in the built environment. Reliable diagnostic tools were developed using two PCR-based molecular typing techniques, one for routine diagnosis and another one for community inventories. Together they provided useful data for characterising the complexity of wood-decay ecosystems and helped reveal the coexistence of different wood-decay fungi within the same microbiotope.     

Application of ion mobility spectrometry to the rapid screening of methamphetamine incorporated in hair

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. Teh detection limit of MA in hair was 0.5 ng mg⁻¹. This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Influence of Pleurotus ostreatus inoculation on wood degradation and fungal colonization

The influence of Pleurotus ostreatus inoculation on wood degradation and on fungal community structure was studied. The experiments were performed on an organically poor fly ash deposit covered with a 10 cm layer of beech wood chips inoculated with P. ostreatus isolate ZIM76. Compared to non-inoculated wood chips, inoculation increased the temperatures and relative humidities and, in the first 6 months, accelerated Klason lignin degradation by 9% and also, after 17 months, increased iron translocation into wood chips by 30%. After 6 months, PCR-DGGE showed 22-28 and 13-21 fungal taxa in non-inoculated and P. ostreatus-inoculated beech chips, respectively. The differences in number of taxa and in the fungal community structure (based on Dice coefficient) between non-inoculated and inoculated wood chips diminished with time. The results indicate that the naturally occurring processes of wood degradation are as efficient as those occurring in sites inoculated with P. ostreatus.     

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43-46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.     

Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide.     

Entomotoxic effects of fungal lectin from Rhizoctonia solani towards Spodoptera littoralis

The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival of an economically important caterpillar in agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for feeding experiments with cotton leafworm. Rearing of insects on a diet containing different concentrations of RSA exerted a strong effect on the larval weight gain. This effect was visible at the lowest concentration of 0.1 % RSA at day 8 and day 11. Interestingly with 1 % RSA, there was a dramatic reduction in larval weight of 89 % at the end of L6 which was followed by a high mortality rate of 82 % in the treated larvae. Furthermore, the other developmental stages of pupation and adult formation were also affected. In addition, the data demonstrated that the combination of RSA with Bt toxin yielded synergistic effects. For instance, 0.03 % RSA+0.005 % Bt toxin caused reduced growth rate and higher mortalities. These findings suggest that RSA is an interesting tool that can be used for bioengineering insect resistance in important agronomical crops.     

Detection of fungal spores from contaminated surfaces by the polymerase chain reaction

A method has been developed to detect fungal spores in dust samples collected from internal surfaces of air-conditioning ducts. The method is based on the utilization of the polymerase chain reaction (PCR) with specific primers for fungal species. PCR amplification is carried out directly in boiled samples avoiding time-consuming DNA preparation steps. The presence of bovine serum albumin in the reaction mixture overcame the inhibitory effect of the humic acids present in the dust.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry

Nanoelectrospray ionization-mass spectrometry and ion mobility-mass spectrometry have been used to study the interactions of the large, multidomain, and conformationally flexible deubiquitinating enzyme ubiquitin specific protease 5 (USP5) with mono- and poly-ubiquitin (Ub) substrates. Employing a C335A active site mutant, mass spectrometry was able to detect the stable and cooperative binding of two mono-Ub molecules at the Zinc-finger ubiquitin binding protein (ZnF-UBP) and catalytic site domains of USP5. Tetra-ubiquitin, in contrast, bound to USP5 with a stoichiometry of 1 : 1, and formed additional interactions with USP5''s two ubiquitin associated domains (UBAs). Charge-state distribution and ion mobility analysis revealed that both mono- and tetra-ubiquitin bound to the compact conformation of USP5 only, and that tetra-ubiquitin binding was able to shift the conformational distribution of USP5 from a mixture of extended and compact forms to a completely compact conformation.     

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

Characterization of the reaction products of cytochrome c with glutathione by mass spectrometry

Cytochrome c and glutathione (GSH) are two important biomolecules that regulate many cellular processes. The reaction of cytochrome c with GSH involves radical oxygen species and exhibits significant complexity. In the present work, the reaction of cytochrome c with GSH in water was characterized using mass spectrometry. The results show for the first time that the reaction generates multiple products including apocytochrome c in oxidized and reduced forms, glutathionylated apocytochrome c, GSH-modified cytochrome c, and oxidized and hydroxylated species. The reaction is O(2) dependent and is rapid in water at neutral pH and 37 degrees C. The reaction involves the cleavage of thioether linkages between the heme and apocytochrome c. Evidence for the role of H(2)O(2) and other oxygen radicals in this reaction is also provided.     

Discovering neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatography and mass spectrometry

Completion of the Caenorhabditis elegans genome sequencing project in 1998 has provided more insight into the complexity of nematode neuropeptide signaling. Several C. elegans neuropeptide precursor genes, coding for approximately 250 peptides, have been predicted from the genomic database. One can, however, not deduce whether all these peptides are actually expressed, nor is it possible to predict all post-translational modifications. Using two dimensional nanoscale liquid chromatography combined with tandem mass spectrometry and database mining, we analyzed a mixed stage C. elegans extract. This peptidomic setup yielded 21 peptides derived from formerly predicted neuropeptide-like protein (NLP) precursors and 28 predicted FMRFamide-related peptides. In addition, we were able to sequence 11 entirely novel peptides derived from nine peptide precursors that were not predicted or identified in any way previously. Some of the identified peptides display profound sequence similarities with neuropeptides from other invertebrates, indicating that these peptides have a long evolutionary history.     

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]⁺ and [PC (18:1/16:0) + Ag]⁺} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.     

Electron-capture dissociation and ion mobility mass spectrometry for characterization of the hemoglobin protein assembly

Native spray has the potential to probe biophysical properties of protein assemblies. Here we report an investigation using both ECD top-down sequencing with an FTICR mass spectrometer and ion mobility (IM) measurements on a Q-TOF to investigate the collisionally induced unfolding of a native-like heterogeneous tetrameric assembly, human hemoglobin (hHb), in the gas phase. To our knowledge, this is the first report combining ECD and ion-mobility data on the same target protein assembly to delineate the effects of collisional activation on both assembly size and the extent and location of fragmentation. Although the collision-induced unfolding of the hemoglobin assembly is clearly seen by both IMMS and ECD, the latter delineates the regions that increasingly unfold as the collision energy is increased. The results are consistent with previous outcomes for homogeneous protein assemblies and reinforce our interpretation that activation opens the structure of the protein assembly from the flexible regions to make available ECD fragmentation, without dissociating the component proteins.     

Effect of fungal infection on the reproductive potential of aphids and their progeny

The effect of infection by Pandora neoaphidis and Beauveria bassiana on the reproductive potential of the pea aphid, Acyrthosiphon pisum, and their progeny was assessed. Infection by either P. neoaphidis or B. bassiana reduced the number of nymphs produced within 24 h of inoculation and over the entire infection period compared to uninfected aphids. However, infection by either P. neoaphidis or B. bassiana for 24 or 72 h did not alter the intrinsic rate of increase of the host aphid's progeny. Therefore, fungal infection appears to have no indirect effects on the fitness of the host's progeny.     

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.     

Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.