MALDI-TOF MS-based analysis of dried seed proteins immobilized on filter paper

Abstract

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.

Discrimination between Eggs from Stink Bugs Species in Europe Using MALDI-TOF MS

“…MALDI-TOF MS is a rapid and simple method, with generally low reagent costs ([ 16 ], Discussion section), or the analysis of protein-containing samples but which requires relatively fresh biological material because the analyzed proteins must not have undergone significant degradation—one of the few limitations of the method. Reeve and Buddie originally reported a convenient and cheap filter-paper-based method for the storage without degradation of plant proteins for later MALDI-TOF MS analysis [ 19 ], which has subsequently been adapted for use with seeds [ 20 , 21 ] and insects [ 22 ] and in the current paper, we have successfully adapted this methodology for use with the eggs of stink bugs (with the ambient-temperature shipping of the samples immobilized on filter paper from Switzerland to the UK after preparation and storage). Once again, as described in detail the Discussion section of [ 22 ], we have used an approach for high-resolution spectral comparisons that departs slightly from the standard Bruker ‘MSP’ method, and we have again used multiple sample replicates (rather than pipetting and/or laser-shot replicates) to accommodate better the real-life variances in the samples and analytical steps.…”

“…To overcome this limitation, Reeve and Buddie, have developed a simple and inexpensive filter-paper-based method for the practical storage of field-sample proteins [ 19 ]. This was originally developed for use with plant material but has since been extended in scope to cover seeds [ 20 , 21 ] and insects [ 22 ]. In each of these method variants, the underlying rationale is to immobilize the proteins from lysed cells onto filter paper by drying thoroughly such that the proteins of interest and any proteases will remain spatially separated while dry.…”

Seed protein biotyping in Amaranthus species: a tool for rapid identification of weedy amaranths of concern

“…Using MALDI-TOF MS spectra from acid-soluble proteins, four different I. glandulifera biotypes differing in susceptibility to a rust biological control agent, were identified [ 52 ]. Extraction of proteins from seed material [ 51 , 52 ], seemed to contain a more stable protein fraction compared to tissue undergoing development (e.g., young leaves) [ 50 ]. Further work studied protein biotyping of tomato varieties, showing good reproducibility for the technique, but low accuracy in distinguishing the test varieties [ 53 ].…”

Seed protein biotyping in Amaranthus species: A tool for rapid identification of weedy amaranths of concern

“…Using MALDI-TOF MS spectra from acid-soluble proteins, four different I. glandulifera biotypes differing in susceptibility to a rust biological control agent, were identi ed [52]. Extraction of proteins from seed material [51,52], seemed to contain a more stable protein fraction compared to tissue undergoing development (e.g., young leaves) [50]. Further work studied protein biotyping of tomato varieties, showing good reproducibility for the technique, but low accuracy in distinguishing the test varieties [53].…”

“…Recently, some research has been developed to test protein biotyping in plants. Importantly, work was performed to distinguish the Impatiens glandulifera from other species in the genus, and also to distinguish its regional biotypes both from leaves and seeds [50][51][52]. Using MALDI-TOF MS spectra from acid-soluble proteins, four different I. glandulifera biotypes differing in susceptibility to a rust biological control agent, were identi ed [52].…”