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

Abstract

BackgroundWe 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).ResultsPeak-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.ConclusionsMALDI-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.Electronic supplementary materialThe online version of this article (10.1186/s13007-019-0412-1) contains supplementary material, which is available to authorized users.

MALDI-TOF MS-Based Analysis of Seed Proteins from Catalogue Varieties of Solanum lycopersicum/Lycopersicon esculentum

“…It is interesting to compare the above with previous studies focused on the invasive weed I. glandulifera, where discrimination between regional biotypes within the species has been demonstrated using both leaf and seed material [10,11], despite working with field-sourced materials. For I. glandulifera seeds collected from the field at four different sites within the UK, the CV for the combined MALDI-TOF MS, sample-preparation, and seed-to-seed variance was 7.539% (largely because seeds collected derive from a wild population of plants growing under uncontrolled conditions) but, for all inter-biotypical comparisons, the average Bruker score was 1.530, with a standard deviation of 0.327.…”

“…From the above, MALDI-TOF MS-based 'fingerprinting' of acid-soluble proteins has been selected because inexpensive and rapid methods have been developed for sample preparation [9] that can discriminate, using leaf samples, between closely-related species and between regional biotypes within a species [10]. Similar discrimination has also been demonstrated using seed material [11]. Given such resolving power, in combination with inexpensive and cheap sample-preparation methodology, an interesting and potentially-beneficial extrapolation was to investigate whether MALDI-TOF MS-based 'fingerprinting' of acid-soluble proteins can be applied to the analysis of plant seeds that result from intensive commercial plant-breeding activity, with the intention of investigating whether this could eventually provide easily-derived and inexpensive molecular-weight markers that could perhaps be linked to beneficial traits, thereby potentially enabling seed selection on this basis.…”

“…For the MALDI-TOF MS analysis of seed material, Reeve and Pollard [11] describe the extraction of acid-soluble seed proteins in 100 µL of acetonitrile/TFA/water/matrix, followed by further dilution (ideally 20-fold to 100-fold) in the same reagent in order to obtain high-quality spectra. In the current article, this method has been slightly modified, with the initial extraction carried out in (50% (v/v) acetonitrile, 2.5% (v/v) TFA, and 47.5% (v/v) water), which is readily available pre-formulated, and which reduces the usage of MALDI matrix.…”

“…glanduliferae). Still with a focus on optimizing the matching of biological control agents to susceptible (regional) target-plant biotypes but extending the scope of the method to include seed material, Reeve and Pollard [11] have also developed a simple and inexpensive method that generates highly-reproducible peak-rich MALDI-TOF MS spectra of acid-soluble seed proteins, and have been able to discriminate successfully between the same four regional biotypes of I. glandulifera. This latter method extracts acid-soluble seed proteins in 100 µL of acetonitrile/TFA/water/matrix, followed by further dilution (ideally 20-fold to 100-fold) in the same reagent in order to obtain high-quality spectra.…”

Spectral Grouping of Nominally Aspergillus versicolor Microbial-Collection Deposits by MALDI-TOF MS

“…In addition to the above, Reeve et al [12] have developed a broadly-applicable yet simple and inexpensive sample-preparation method that lyses microbial cells by immersion in aqueous acetonitrile that also contains trifluoroacetic acid (TFA) to extract acid-soluble proteins, along with near-saturated and inexpensive-grade α-cyano-4-hydroxycinnamic acid (HCCA) matrix. This method can also be applied to insect [12], plant, [12,13], and seed material [14] simply by macerating the biomass of interest in the above reagent. After immersion (or maceration), the resulting matrix-saturated lysate, which also contains the extracted proteins for sample characterization or identification, is simply dried directly onto the MALDI-TOF MS sample plate ready for analysis.…”