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In the current paper, we used a method based on stink bug egg-protein immobilization on filter paper by drying, followed by post-(storage and shipping) extraction in acidified acetonitrile containing matrix, to discriminate between nine different species using MALDI-TOF MS. We obtained 87 correct species-identifications in 87 blind tests using this method. With further processing of the unblinded data, the highest average Bruker score for each tested species was that of the cognate reference species, and the observed differences in average Bruker scores were generally large and the errors small except for Capocoris fuscispinus, Dolycoris baccarum, and Graphosoma italicum, where the average scores were lower and the errors higher relative to the remaining comparisons. While we observed clear discrimination between the nine species using this method, Halyomorpha halys and Piezodorus lituratus were more spectrally related than the other pairwise comparisons.
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Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry can be used for the characterization and identification of filamentous fungi, for which it is desirable to have a means of growth in which the resulting spectra remain as consistent as possible over time. To this end, we initially opted for growth in oil-overlaid small-volume liquid culture, using a medium (Czapek Dox) not containing significant amount of proteins or peptides, and with protein extraction from the entire culture volume. For both 3-week and 10-day time courses, however, we observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Guided by the above, we next employed a more nutrient-rich MALDI-TOF MS-compatible liquid-culture medium, now used without an oil overlay. For a 10-day time course, we again observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Finally, we opted for a method employing filter-paper-supported growth in the same MALDI-TOF MS-compatible rich medium within sealed 1.5 ml Eppendorf tubes, again with protein extraction from the entire culture volume. Using this final method, while we observed significant spectral changes between 2 days and 3 days, from 3 days to 10 days the spectra remained very consistent, with comparable peak richness to agar-plate controls. This method gave slightly better identifications and lower spectral variance compared to agar-plate controls, and the use of this method for the construction of growth-time-point-specific databases for fungal identification is discussed.
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Analytical techniques currently available for the characterization of mixtures of microorganisms are generally based on next-generation sequencing. Motivated to develop practical and less-expensive methods for characterizing such mixtures, we propose, as an alternative or complement, the use of matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is capable of high-resolution discrimination between species and even between biotypes within species. Potential approaches employing this technique for such characterization are discussed along with impediments to their successful employment. As a consequence, our rationale has been to capitalize on the powerful algorithms currently available for spectral comparison. Following this rationale, the first priority is to ensure the generation of MALDI-TOF MS spectra from mixtures of microorganisms that contain manageable peak complexities and that can be handled by the existing spectral comparison algorithms, preferably with the option to archive and re-run sample preparations and to pipette replicates of these onto MALDI-TOF MS sample plates. The second priority is to ensure that database entry is comparably facile to sample preparation so that large databases of known microorganism mixture MALDI-TOF MS spectra could be readily prepared for comparison with the spectra of unknown mixtures. In this article, we address the above priorities and generate illustrative MALDI-TOF MS spectra to demonstrate the utility of this approach. In addition, we investigate methods aimed at chemically modulating the peak complexity of the obtained MALDI-TOF MS spectra.
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Matrix-assisted laser-desorption and ionization time-of-flight mass spectroscopy (MALDI-TOF MS) is commonly used for the characterization of protein-containing biological samples. For this, we have previously developed sample-preparation methods that can be used for discrimination between Impatiens species and also between regional biotypes of Himalayan balsam (Impatiens glandulifera), initially using leaf samples and, more recently, using seed material. In the current article, we have developed a further MALDI-TOF MS-based method that can be used with seeds that uses only simple equipment and minimally hazardous reagents prior to storing and/or shipping dried seed proteins immobilized on filter paper for MALDI-TOF MS analysis. We have investigated I. glandulifera regional-biotype seeds originating from four different sites within the UK for which the parent plants differ in their susceptibility to the biological control agent Puccinia komarovii var. glanduliferae. Using a combination of time-course comparisons and principal-component analysis, we have demonstrated good MALDI-TOF MS spectral conservation, even after storage for 1 month at 35°C, of dried seed-protein samples immobilized on filter paper. This method may provide a further useful tool for the matching of biological control agents optimally to susceptible (regional) target-plant biotypes, and for seed characterization and/or identification in general.
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The fruit fly Drosophila suzukii has recently become an invasive pest insect of significant economic impact in Europe and the USA. In contrast to other Drosophila species, D. suzukii is able to infest intact fruit by means of a saw-like ovipositor, which allows females to deposit eggs beneath the skin of the fruit. Classical biological control using the parasitoid wasp Ganaspis cf. brasiliensis is currently being researched as an environmentally sustainable option for the control of D. suzukii. In particular, the host specificity of this parasitoid has been assessed for populations from different regions in China and Japan. In order to study the relationship between the differences in specificity and molecular variations, we have adapted a matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method, originally developed for use with plant material, to discriminate between example populations of G. cf. brasiliensis. We have employed a combination of principal component analysis and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect protein spectra generated, between four populations of G. cf. brasiliensis (originally collected from Tokyo and Hasuike in Japan and Dali and Ximing in China). MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations G. cf. brasiliensis. The Chinese populations were observed to be similar, but the Tokyo population is slightly different and the Hasuike population is significantly different from the other populations. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population, even though both originate from Japan.
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BACKGROUND: We have recently developed a simple, rapid, and relatively-cheap method for matrix-assisted laser-desorption and ionisation time-of-flight mass spectroscopy (MALDI-TOF MS) sample preparation that is applicable to plant material (in addition to microbial and insect material), and have used this to discriminate between closely-related Impatiens species and between regional biotypes of the invasive weed Impatiens glandulifera (commonly known as Himalayan balsam) using leaf samples. In the current paper, we have developed a complementary MALDI-TOF MS-based method for use with seeds. We have employed a combination of principal-component analysis and blind-tested comparison between reference-sample MALDI-TOF MS spectra and test-sample spectra to discriminate, on the basis of the acid-soluble seed-protein spectra generated by our method, between four regional biotypes of I. glandulifera from within the UK that differ in their susceptibility to the biological control agent Himalayan balsam rust (Puccinia komarovii var. glanduliferae). RESULTS: Peak-rich and highly-reproducible spectra were obtained and, in blind testing with test seeds collected in 2017 against reference seeds collected in 2017, we observed 100% identification accuracy in 12 blind tests. In blind testing with test seeds collected in 2016 against reference seeds collected in 2017, we observed 92% identification accuracy in 12 blind tests. CONCLUSIONS: MALDI-TOF MS analysis of seed material is able to discriminate between regional biotypes of I. glandulifera. MALDI-TOF MS therefore has the potential to improve the efficiency and efficacy of weed biological control using co-evolved natural enemies of invasive non-native plant species, through the matching of biological control agents with susceptible regional biotypes.
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In matrix-assisted laser-desorption and ionization mass spectrometry, spectral differences are frequently observed using different growth media on agar plates and/or different growth times in culture, which add undesirable analytical variance. In this article, we explore an approach to the above problem based upon the rationale that, while protein expression in fungal mycelium may well vary under different growth conditions, this might not apply to the same extent in fungal spores. To this end, we have exploited the fact that while mycelium is generally anchored to the fungal-growth substrate, some fungi produce physically-isolated spores which, as such, are amenable to manipulation using dielectrophoresis (the translational motion of charged or uncharged matter caused by polarization effects in a non-uniform electrical field). Such fields can be conveniently generated through the charging of an insulator using the triboelectric effect (the transfer of charge between two objects through friction when they are rubbed together). In this study, polystyrene microbiological inoculating loops were used in combination with nylon-fabric rubbing to harvest fungal spores from five species from within the genus Penicillium, which were grown on agar plates containing two different media over an extended time course. In terms of average Bruker spectral-comparison scores, our method generated higher scores in 80% of cases tested and, in terms of average coefficients of variation, our method generated lower spectral variability in 93% of cases tested. Harvesting of spores using a rapid, inexpensive and simple dielectrophoretic method, therefore, facilitates improved fungal identification for the Penicillium species tested.
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Historical microbial collections often contain samples that have been deposited over extended time periods, during which accepted taxonomic classification (and also available methods for taxonomic assignment) may have changed considerably. Deposited samples can, therefore, have historical taxonomic assignments (HTAs) that may now be in need of revision, and subdivisions of previously-accepted taxa may also be possible with the aid of current methodologies. One such methodology is matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). Motivated by the high discriminating power of MALDI-TOF MS coupled with the speed and low cost of the method, we have investigated the use of MALDI-TOF MS for spectral grouping of past deposits made to the Centre for Agriculture and Bioscience International (CABI) Genetic Resource Collection under the HTA Aspergillus versicolor, a common ascomycete fungus frequently associated with soil and plant material, food spoilage, and damp indoor environments. Despite their common HTA, the 40 deposits analyzed in this study fall into six clear spectral-linkage groups (containing nine, four, four, four, four, and two members, respectively), along with a group of ten spectrally-unique samples. This study demonstrates the clear resolving power of MALDI-TOF MS when applied to samples deposited in historical microbial collections.
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Key chili and maize growing areas of Pakistan were selected for a focused baseline study of the levels of Aspergillus spp. Investigations were undertaken using a combination of molecular and culture-based techniques. Samples investigated included soil samples, one-year-old corn cobs, and fresh chili from selected locations. Aspergillus strains obtained from corn cobs were screened using coconut milk agar, resulting in one strain that was positive for aflatoxin production. Whole genome sequencing (WGS) with low coverage techniques were employed to screen the isolates for differences in the ribosomal RNA gene cluster and mitochondrial genome, with the aflatoxigenic strain proving to have a distinctive profile. Finally, strains were subjected to matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in order to obtain a proteomic 'fingerprint' which was used to distinguish the aflatoxigenic strain from the other isolates. The next generation sequencing (NGS) study was broadened to incorporate metabarcoding with ITS rRNA for determining the microbial biodiversity of the soil samples and presumptive screening for the presence of aflatoxigenic strains. Using information gleaned from the WGS results, a putative aflatoxigenic operational taxonomic unit (OTU) was observed in four of the 15 soil samples screened by metabarcoding. This method may have beneficial applications in early detection and surveillance programs in agricultural soils and commodities.
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BACKGROUND: Protein-containing samples can readily be characterised and/or identified using matrix-assisted laser-desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). This technique however requires relatively-fresh biological material that contains proteins that have not yet undergone significant degradation. For field-work collection of samples, problems are often encountered due to delays between collection and sample processing, sample storage (possibly at elevated temperature and/or humidity in some climates), quarantine/regulatory restrictions on the transfer of living biological materials across national borders, and the potential to transfer unwanted microorganisms via non-living biological materials. RESULTS: In an attempt to overcome the above difficulties, we have developed a simple and inexpensive method for practical storage of field-sample proteins, for subsequent MALDI-TOF MS analysis, in which biological material is crushed onto filter paper and dried. The dried and protein-impregnated filter paper can then be soaked in an alcoholic solution suitable for the inactivation of microorganisms of concern and again dried for storage. After subsequent dry storage, the proteins may be eluted from the paper using a solution containing acetonitrile, trifluoroacetic acid, water, and MALDI-TOF MS matrix near to saturation. The extracted proteins are then pipetted onto the MALDI-TOF MS sample plate for subsequent analysis. Using this method, spectra of comparable quality to fresh-material controls have been obtained for acid-soluble proteins from Fallopia japonica and Impatiens glandulifera leaf material. Unlike untreated leaf material, high-quality spectra can be obtained with and without alcohol treatment even after storage for one month at up to 40 °C. CONCLUSIONS: We have developed a simple and inexpensive method for practical storage of field-sample proteins for subsequent MALDI-TOF MS analysis. Key benefits of this approach are a reduction in sample degradation, and consequent conservation of taxon-discriminatory spectral profiles, whilst minimising the potential for carryover of viable microorganisms.
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BACKGROUND: Matrix-assisted laser-desorption and ionisation time-of-flight mass spectroscopy (MALDI-TOF MS) is a powerful tool for the characterisation and/or identification of protein-containing samples. Several MALDI-TOF MS sample-preparation methods are currently available but few of these are well suited to the analysis of plant material. We have recently developed a simple, rapid, and relatively-cheap method for MALDI-TOF MS that is applicable to plant material (in addition to microbial and insect material), and our aim in this study was to distinguish between closely-related plant species and/or between regional biotypes within an invasive weed species using this method with a view to optimising the selection of biological control agents that can be used for weed management. RESULTS: We have employed a combination of principal-component analysis and closest-relatedness diagrams derived from MALDI-TOF MS spectral-comparison data to discriminate between the closely-related Impatiens spp. Impatiens noli-tangere, Impatiens parviflora, Impatiens scabrida, Impatiens balsamina, and two regional biotypes of the invasive weed Impatiens glandulifera. We have also developed a method for sample discrimination based upon comparison between blind-test MALDI-TOF MS spectra and reference-sample spectra. Using this latter method, we have been able to discriminate on the basis of the acid-soluble-protein mass spectra generated between four regional biotypes of I. glandulifera that differ in their susceptibility to the biological control agent Himalayan balsam rust (Puccinia komarovii var. glanduliferae) using mature leaf material. Using younger leaves, discrimination was not possible between these four regional biotypes. CONCLUSIONS: MALDI-TOF MS analysis is able to discriminate between closely-related plant species within the genus Impatiens and between regional biotypes of I. glandulifera. Because of this, MALDI-TOF MS holds great promise for improving weed biological control, a management technique which uses highly-specific co-evolved natural enemies for the control of an invasive non-native plant species, through the optimal matching of biological control agents with susceptible target species/regional biotypes.
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Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry prepares proteins intact in the gas phase with predominantly a single positive charge. The times-of-flight of charged proteins along a tube held at high vacuum after acceleration in an electrical field are proportional to the square root of the mass-over-charge ratios for the proteins, thereby allowing a mass spectrum to be generated, which can then be used to characterize or identify a protein-containing sample. Several sample-preparation methods are currently available but not all of these are applicable to some forms of fungal biomass and few of these are well suited to the analysis of plant or insect material. We have therefore developed a simplified method that: lyses cells, selectively solubilizes basic proteins, dissolves matrix to a suitable concentration, generates spectra with good intensity and peak richness, costs no more (and generally less) than current methods, and is not constrained in terms of throughput by the availability of centrifuges. Using this method, and a reagent formulation comprising α-cyano-4-hydroxycinnamic acid matrix close to saturation in 60%-65% (v/v) acetonitrile in water containing 2.5% (v/v) trifluoroacetic acid, we have been able to differentiate between strains for a representative subset of aflatoxin-producing and aflatoxin-non-producing strains of Aspergillus fungi, to differentiate between Indian and Pakistani strains of Himalayan balsam rust, to differentiate between closely-related Crassula spp. and regional biotypes of Crassula helmsii, and to differentiate between rubbervine introduced into Australia and Brazil. We have also analyzed fall armyworm and stem-borer samples stored in 70% (v/v) ethanol and old dried insect specimens.