Proteomic analysis of the distribution of the major seed allergens in wild,landrace, ancestral,and modern soybean genotypes

BACKGROUND: A comparative analysis of seed allergens from various soybean genotypes is crucial for identifying and eliminating potential allergens. We have investigated the distribution of three major allergens (Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K) in wild, landrace, ancestral and modern soybean genotypes. RESULTS: Gly m Bd 60K allergens consist of α subunits of β‐conglycinin and G2 subunits of glycinin. In wild genotypes, α subunits of β‐conglycinin separated into six to seven protein spots whereas five to seven spots were observed in the landraces. All genotypes of modern and ancestral groups showed 3–5 protein spots of α subunits of β‐conglycinin. All genotypes showed eight spots of glycinin G2 subunits except one ancestral genotype which had seven spots. Two protein spots were detected for Gly m Bd 30K in 14 genotypes but one spot was detected in two wild genotypes. Two protein spots were detected for Gly m Bd 28K in all genotypes. CONCLUSION: Considerable heterogeneity of the α subunit of β‐conglycinin distribution exists among these 16 soybean genotypes. Significant proteomic variation was observed between different soybean groups rather than among genotypes in the same group. This investigation would be valuable to researchers working with soybean and nutrition. Copyright © 2007 Society of Chemical Industry     

高效降解棉酚菌种的筛选及棉粕发酵脱毒工艺研究

通过醋酸棉酚YPG培养基多级逐步驯化筛选,结合棉粕固态生物发酵的方法,筛选出1株高效降解棉酚的热带假丝酵母JD-9。通过单因素试验初步确定了棉粕发酵脱毒的工艺条件,进一步利用分式析因试验设计法,对影响棉粕脱毒率的因素进行了筛选,方差分析表明,影响棉粕发酵脱毒的主要因素为热带假丝酵母JD-9接种量和卡氏酵母JD-13接种量。通过响应面分析法和典型性分析得出棉粕发酵脱毒的最优条件为:热带假丝酵母JD-9接种量4.42%,卡氏酵母JD-13接种量0.56%,发酵时间48 h,发酵温度30℃,料水比1∶0.8,棉粕中的棉酚从987.5 mg/kg降解至85.9 mg/kg,脱毒率达到91.3%。     

Molecular cloning,structural analysis and mass spectrometric identification of native dioscorins of various yam species

BACKGROUND: Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and elucidated which dioscorin isoforms are the major constituents in tubers. RESULTS: The dioscorin gene families of D. japonica (Dj‐dioA1‐Dj‐dioA4, Dj‐dioB1 and Dj‐dioB2) and D. pseudojaponica (Dp‐dioA1‐Dp‐dioA5 and Dp‐dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da‐dioscorins), D. japonica (Dj‐dioscorins) and D. pseudojaponica (Dp‐dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys²⁸‐Cys¹⁸⁷) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys⁴⁰‐Cys⁴⁰). Results of MALDI‐TOF‐MS revealed that the Da‐dioscorins were mainly encoded by Da‐dioA2, Da‐dioA3 and Da‐dioA4. The majority of the Dj‐dioscorins were encoded by Dj‐dioA1, Dj‐dioA2, Dj‐dioA3 and Dj‐dioB2. The Dp‐dioscorins mainly comprised proteins encoded by Dp‐dioA1, Dp‐dioA3, Dp‐dioA4, Dp‐dioB1 and Dp‐dioB2. CONCLUSION: Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions. © 2012 Society of Chemical Industry     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

烤烟有益真菌菌株的筛选与鉴定

为比较不同真菌对烟叶品质的影响,用分离到的27株真菌菌株处理烟叶,通过烟叶化学成分和评吸结果的比较得出:SZ14和YZ2 2个菌株处理烟叶后,烟叶化学成分较协调,烟叶评吸得分明显高于对照;CZ3、CZ12、CZ19、CZ22、CZ61、CZ62和SZ5 7个菌株处理烟叶后,烟叶化学成分较协调,评吸得分高于对照;其它18个菌株对烟叶品质影响不明显。对得分较高的2个菌株进行分子鉴定表明,YZ2为木霉属长梗木霉(Trichoderma longibrachiatum),SZ14为曲霉属(Aspergillussp)。     

游离棉酚脱除菌株的分离鉴定及体内定植效果研究

该研究从藜麦茎叶和番茄杆样品中筛选分离出高效游离棉酚脱除菌株,为棉籽粕生物脱毒提供优良菌株。测定筛选菌株的游离棉酚脱除能力及产蛋白酶能力,采用人工胃液、人工肠液耐受实验,抗生素敏感实验及表面特性实验研究筛选菌株在体内定植能力,并通过形态学观察、生理生化试验及16S rDNA序列分析对筛选菌株进行鉴定。结果表明,筛选出1株高效游离棉酚脱除芽孢杆菌,编号为S-77,其游离棉酚脱除率为81.30%,产蛋白酶酶活为767.27 U/g。菌株S-77被鉴定为东洋芽孢杆菌（Bacillus toyonensis）,具有耐人工胃液、肠液效果及较好黏附于宿主肠壁细胞的能力,对抗生素敏感,可用于棉籽粕生物脱毒。     

Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry

Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.     

Surfome analysis of a wild-type wine Saccharomyces cerevisiae strain

The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry.     

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE‐mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX‐4T‐3 and expressed in Escherichia coli BL 21. 2‐D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies. Copyright © 2011 Society of Chemical Industry     

Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation

Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an ‘‐omics’ level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen‐limited medium and performed a comparative and semi‐quantitative proteomic analysis of cell samples obtained thereafter by a 2D‐LC–MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi‐quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non‐filamentous conditions

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface‐disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild‐type strain. The strains were grown under filamentous and non‐filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI–TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild‐type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI–TOF MS is a reliable technique in identifying mutant‐specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of chicken,duck, and quail egg yolks

A comparative analysis of egg yolk proteome of three major poultry species, namely, chicken, duck, and quail, was carried out with two-dimensional (2D) gel electrophoresis patterns and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Proteins identified from these three poultry species shared high degrees of sequence and structure homology, which might be related to the bird’s adaptability under different environmental stresses. Few specific proteins were found in this study. Comparative 2D gel electrophoresis patterns of chicken, duck, and quail revealed that some protein-specific regions on gels could be used for authentic identification of poultry eggs. These findings could provide a fundamental understanding of different poultry egg protein profiles and showed us some new sights in egg yolk nutrients related to certain properties and functions.