Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol

Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; β-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems.

Application of Parallel Reaction Monitoring in 15N labeled Samples for Quantification

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

Avenanthramides: Unique Bioactive Substances of Oat Grain in the Context of Cultivar, Cropping System, Weather Conditions and Other Grain Parameters

Our study was focused on the evaluation of the content of a wider spectrum of eight avenanthramides (AVNs) as unique components of oat grain under the effects of four selected factors (cultivar, locality, cropping system, and year). The weather effects on changes in the AVN content and their relationship to other important parameters of oat grain were further evaluated in more detail. A sensitive UHPLC system coupled with a QExactive Orbitrap mass spectrometer was used for AVN quantification. AVNs confirmed a high variability (RDS = 72.7–113.5%), which was dominantly influenced by the locality and year factors. While most AVN types confirmed mutually high correlations (r = 0.7–0.9), their correlations with the other 10 grain parameters were lower (r ≤ 0.48). Their significant correlations (0.27–0.46) with β-D-glucan could be used in perspective in breeding programs for the synergetic increase of both parameters. PCA analysis and Spearman correlations based on individual cultivars confirmed a significant effect of June and July precipitation on the increase of Σ AVNs. However, the results also indicated that higher precipitation can generate favorable conditions for related factors, such as preharvest sprouting evoking a direct increase of AVNs synthesis in oat grain.

Metabolomic Changes in Naturally MAP-Infected Holstein–Friesian Heifers Indicate Immunologically Related Biochemical Reprogramming

Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein–Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~ 1 to 19 months of age. Sera were assessed using flow infusion electrospray–high-resolution mass spectrometry (FIE–HRMS) on a Q Exactive hybrid quadrupole–Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP.

QuEChERS-Based Methodology for the Screening of Alkylphenols and Bisphenol A in Dairy Products Using LC-LTQ/Orbitrap MS

A simple methodology was developed for the determination of four Endocrine Disrupting Chemicals (EDCs) in dairy products. The EDCs included alkylphenols (4-tert-octylphenol, technical nonylphenol isomers, 4-nonylphenol) and bisphenol-A. The methodology consisted of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction followed by liquid chromatography (LC) coupled to the hybrid LTQ/Orbitrap mass spectrometer (MS). The high resolution (HR) analysis provided the required selectivity demonstrating excellent sensitivity and enabled the high-mass accuracy of the analytes within short time of analysis, after a chemometric optimization of the instrument parameters. An experimental design was employed for the estimation of the effect of different parameters on the QuEChERS extraction efficiency to obtain the optimum conditions. Method validation proved that analysis exhibited excellent linearity (R2 > 0.9966), low enough precision (0.6 to 13.3%) and recoveries in the range of 91 to 108%. Limits of detection (LOD < 6.5 ng g−1) and quantification (LOQ < 20 ng g−1) as well as matrix effects (ME) were also evaluated. The optimized method was successfully applied to analyze dairy commodities varying in fat content and packaging material including milk, yogurts and infant formulae. Detected concentration levels (MDL-10.4 ng g−1) for bisphenol-A BPA in milk samples resulted in 0.36% of TDI for the medium case (average BPA concentrations) and 1.15% of TDI for the worst case (maximum BPA concentration).