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Discrimination between regional biotypes of Impatiens glandulifera using a simple MALDI-TOF MS-based method for use with seeds.

ABSTRACT: 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.

SUBMITTER: Reeve MA

PROVIDER: S-EPMC6416845 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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Discrimination between regional biotypes of <i>Impatiens glandulifera</i> using a simple MALDI-TOF MS-based method for use with seeds.

Reeve Michael A MA Pollard Kathryn M KM

Plant methods 20190314

<h4>Background</h4>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 <i>Impatiens</i> species and between regional biotypes of the invasive weed <i>Impatiens glandulifera</i> (commonly known as Himalayan balsam) usi ...[more]

PMID: 30911324

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Project description: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.

Project description:BackgroundProtein-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.ResultsIn 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.ConclusionsWe 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.

Project description:We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.

Dataset Information

Discrimination between regional biotypes of Impatiens glandulifera using a simple MALDI-TOF MS-based method for use with seeds.

Publications

Discrimination between regional biotypes of <i>Impatiens glandulifera</i> using a simple MALDI-TOF MS-based method for use with seeds.

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