RESUMO
Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-to-vacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental set-up and optimization strategy is described for liquid AP-MALDI MS which improves the ionization efficiency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2fmol/µL (0.5µL, i.e. 1fmol, deposited on the target) with very low sample consumption in the low nL-range.
Assuntos
Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Pressão Atmosférica , Íons/química , Peptídeos/química , Proteínas/classificaçãoRESUMO
Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.
RESUMO
We report for the first time the extraction, isolation, and the proposed structure of a polysaccharide from the fruiting bodies of Daedalea quercina. The monosaccharide composition of D. quercina isolate (DQW1Pa1) was mainly glucose as identified using GC-MS. FTIR-ATR spectroscopy and absolute configuration studies showed that this polysaccharide is a ß-d-glucan. Its average molecular weight obtained using size exclusion chromatography was 1.6×104Da, consistent with glucans derived from the order Polyporaceae. MALDI-QTOF MS/MS was carried out to identify the linkage and connectivity of the glucose units. Collision Induced Dissociation (CID) of selected parent ions of different oligosaccharide lengths showed the presence of characteristic glycosidic bond cleavages Bn/Cn, the linear backbone by 1-6 linkage, and the cross-ring fragment, 0,3An. Presence of branching unit was identified from high intensity 0,3A4 fragment and verified from diagnostic ion of [D] and [D-H2O] types. To confirm the linkage assignment obtained using MALDI-QTOF MS/MS, DQW1Pa1 was subjected to methylation analysis. Results showed the presence of 1-3, 1-6, 1- and 1-3-6 linked glucose in the order of decreasing abundance, respectively. The repeating unit of isolate DQW1Pa1 was deduced as 1-3 linked linear glucose backbone with branches composed of three 1-3 linked glucose units connected to backbone by 1-6 linkage.