Rapid screening of Alternaria mycotoxins using MALDI-TOF mass spectrometry.

BACKGROUND: Members of the Alternaria genus produce various toxins whose occurrence in agricultural commodities is a major concern for humans and the environment. The present study developed a simple and efficient matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the rapid detection of Alternaria toxins. RESULTS: A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) by MALDI-TOF MS was developed. Different solid phase extraction (SPE) clean-up methods were tried to optimize the purification of wheat matrix, and an optimal extraction method was designed to recover the three Alternaria toxins. In addition, various MALDI matrices were examined and α-cyano-4-hydroxycinnamic acid (CHCA) matrix gave good repeatability for all three Alternaria toxins. CONCLUSION: This is the first study to report the detection of three important Alternaria toxins concurrently using MALDI-TOF MS and opens up the possibility of rapid screening of Alternaria toxins in several other cereals and food products. © 2016 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2016 Society of Chemical Industry.

Quantitative Proteomics Analysis of Camelina sativa Seeds Overexpressing the AGG3 Gene to Identify the Proteomic Basis of Increased Yield and Stress Tolerance.

Camelina sativa, a close relative of Arabidopsis, is an oilseed plant that is emerging as an important biofuel resource. The genome and transcriptome maps of Camelina have become available recently, but its proteome composition remained unexplored. A labeling LC-based quantitative proteomics approach was applied to decipher the Camelina seed proteome, which led to the identification of 1532 proteins. In addition, the effect of overexpression of the Arabidopsis G-protein γ subunit 3 (AGG3) on the Camelina seed proteome was elucidated to identify the proteomic basis of its increased seed size and improved stress tolerance. The comparative analysis showed a significantly higher expression of proteins involved in primary and secondary metabolism, nucleic acid and protein metabolism, and abscisic acid related responses, corroborating the physiological effects of AGG3 overexpression. More importantly, the proteomic data suggested involvement of the AGG3 protein in the regulation of oxidative stress and heavy metal stress tolerance. These observations were confirmed by the physiological and biochemical characterization of AGG3-overexpressing seeds, which exhibit a higher tolerance to exogenous cadmium in a glutathione-dependent manner. The activity of multiple redox-regulating enzymes is higher in seeds expressing enhanced levels of AGG3. Overall, these data provide critical evidence for the role of redox regulation by the AGG3 protein in mediating important seed-related traits.

Identification of six new Alternaria sulfoconjugated metabolites by high-resolution neutral loss filtering.

RATIONALE: Many species of Alternaria damage important agricultural crops, including small grains and tomatoes. These fungi can produce a variety of secondary metabolites, some of which are toxic to humans and animals. Interest in screening for conjugated or 'modified' mycotoxins has increased because of their tendency to evade traditional analytical screening methods. Two sulfoconjugated Alternaria toxins have been reported and the potential exists for many more. METHODS: One hundred and forty-eight Canadian strains of Alternaria spp., about half of them isolated from grain, were grown on Potato Dextrose Agar in Petri dishes for 7 days. Plugs of each strain were removed, extracted and screened by a rapid liquid chromatography (LC)/data-dependent tandem mass spectrometry (MS(2)) method in negative electrospray ionization mode. Data generated on an Orbitrap Q-Exactive mass spectrometer was processed by post-acquisition neutral loss filtering (NLF). Seven isolates that produced sulfoconjugates of known Alternaria toxins were selected for growth on three additional types of fermentation media. RESULTS: Collision-induced dissociation of sulfoconjugated ions displayed a distinctive neutral loss of SO3 (79.957 Da) that was detected in the MS(2) datasets using post-acquisition NLF. A total of 108 of the 148 isolates screened produced sulfoconjugated metabolites on agar plates. Analysis of the seven isolates grown in liquid culture, on rice and Cheerios, led to the discovery of six new, two previously reported and 30 unidentified sulfoconjugated compounds. CONCLUSIONS: NLF of HRMS(2) data from an Orbitrap Q-Exactive is a powerful tool for the rapid discovery of sulfoconjugated fungal metabolites. This technique could also be applied to the detection of other important conjugated mycotoxins such as glucosides. The majority of the Canadian isolates of Alternaria spp. studied produced sulfoconjugated metabolites, some of which had no known 'free' Alternaria precursor metabolite, indicating that they are possibly new metabolites. The advantage of sulfoconjugation to Alternaria spp. is unknown, and warrants further study into the mechanisms behind the sulfur assimilatory pathways.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase.

Economically viable production of solvents through acetone-butanol-ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose.

BACKGROUND: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetone-butanol-ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. RESULTS: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. CONCLUSION: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.

Rapid Screening of Ergot Alkaloids in Sclerotia by MALDI-TOF Mass Spectrometry.

Ergot is a common disease of wheat and other cereal grains that is predominantly caused by Claviceps purpurea in the field, often affecting crop yield in addition to the environment. Infected grain can be contaminated with dark sclerotia, which contain fungal metabolites such as ergot alkaloids. The occurrence of ergot alkaloids in cereal grain is a major health concern for humans and livestock. Effective and rapid screening of these mycotoxins is crucial for producers, processors, and consumers of cereal-based food and feed grain. Established methods of ergot alkaloid screening based on LC-MS or GC-MS require laborious processes. A novel method using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS was developed to identify four ergot alkaloids. Using dihydroxybenzoic acid as the matrix, ergosine, ergocornine, ergocryptine, and ergocristine were readily detected in individual sclerotia of C. purpurea. The accuracy of the identified ergot alkaloids was further confirmed by tandem MS analysis. MALDI-TOF MS is suitable for high-throughput screening of ergot alkaloids because it permits rapid and accurate identification, simple sample preparation, and no derivatization or chromatographic separation.

ABCB1 genetic polymorphism and risk of upper aerodigestive tract cancers among smokers, tobacco chewers and alcoholics in an Indian population.

BACKGROUND AND OBJECTIVE: Upper aerodigestive tract (UADT) cancers are associated with the tobacco use and alcohol consumption. Certain toxins and carcinogens causing UADT cancers are found to be substrates of polymorphic ABCB1 gene encoded P-glycoprotein efflux pump. This study investigates the association between ABCB1 gene polymorphism at exon 26 (3435C>T) and risk to UADT cancers in Tamilians, a population of south India. METHODS: The study included 219 unrelated histopathologically confirmed cases and 210 population-based controls. Genomic DNA was extracted from peripheral leukocytes and genotyped for ABCB1 3435C>T polymorphism by PCR-restriction fragment length polymorphism method. RESULTS: The multivariate logistic regression analyses demonstrated that the homozygous ABCB1 TT genotype was significantly associated with an overall increased risk for developing UADT cancers [odds ratio (OR): 2.53; 95% confidence interval (CI): 1.28-5.02]. Further, the determination of gene-environment interaction by stratified analyses have revealed a significant interaction between the smoking and homozygous TT genotype [(OR: 7.52; CI: 1.50-37.70) and (OR: 16.89; CI: 3.87-73.79) for 11-20 and >20 pack-years, respectively]. The strongest interaction was observed among the regular tobacco chewers (OR: 45.29; CI: 8.94-130.56) homozygous for TT genotype. No suggestion, however, of an interaction between the genotypes and the alcohol consumption on the multiplicative scale was made. CONCLUSION: The ABCB1 gene polymorphism at exon 26 (3435C>T) may be one of the risk factors for susceptibility to UADT cancers. Furthermore, the significant interaction among habitual smokers and tobacco chewers, homozygous for TT genotype modulates the risk to UADT cancers in the Tamilian population of south India.