Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera)

To compare the protein complement of royal jelly (RJ) from high RJ producing honeybees ( Apis mellifera L.), a strain of A. mellifera artificially selected for increased RJ production from Italian honeybees in China for more than two decades was compared to those of native Italian honeybees ( A. mellifera L.) and Carnica honeybees ( A. mellifera C.); the protein in RJ from these three strains of honeybees was partially identified by using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and a protein engine identification tool applied to the honeybee genome. The results showed that 152, 157, and 137 proteins were detected in the three species of RJ; among which 57, 57, 51 high abundant proteins ere identified, respectively. Most identifited spots, 45, 45, 41, were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. Also, 3-glucose oxidase, 1-peroxiredoxin (PRDX), and 1-glutathione S-transferase (GST) S1 were identified in three RJ samples. Furthermore, during the determination of the peptides mass fingerprinting (PMF) of each spot, for the first time, PRDX and GST S1 proteins have been identified in RJ. Thus, the results suggest that the protein complement of high RJ producing honeybees is not different compared to native Italian honeybees, while a difference remains between Carnica honeybees.     

Novel royal jelly proteins identified by gel-based and gel-free proteomics

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.     

Analysis of Lupinus albus storage proteins by two-dimensional electrophoresis and mass spectrometry

A laboratory-prepared total protein extract (TPE) and a lupin protein isolate (LPI-E) produced in a pilot plant were submitted to a detailed two-dimensional (2DE) proteomic investigation. Recent findings have indicated that in an established rodent model of hyperlipidemia, moderate daily intakes of LPI-Es lead to a reduction of total and low-density lipoprotein cholesterol levels, and the knowledge of the actual composition of the protein sample used in that study is at the basis of further structure/action investigations. The experimental results indicate that the semi-industrial procedure used for the production of LPI-E damages only marginally the proteins. It does, however, cleave some disulfide bridges and induce mild proteolysis, as confirmed by the higher number of resolved protein spots in the low Mr and acidic pI region of the 2DE map. Out of 72 spots submitted to mass spectrometry and compared with available protein databases, 42 correspond to fragments of beta-conglutin, the 7S globulin of lupin, spanning between positions 37 and 495 of the protein sequence. Using the bioinformatic tool BlastP, these peptides were compared to the alpha'-subunit of beta-conglycinin, the 7S globulin of soybean, this being the most active hypocholesterolemic component of soybean protein, as shown by in vitro and in vivo experiments. At least 18 peptides derived from beta-conglutin, having a percentage identity higher than 50% and a similarity percentage higher than 70% vs the alpha'-subunit of beta-conglycinin, are likely candidates to be the biologically active components of lupin protein.     

Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography

To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.     

Characterization of kafirin and zein oligomers by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.     

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.     

One- and two-dimensional gel electrophoresic identification of African yam bean seed proteins

Seed proteins were extracted from the African yam bean (AYB; Sphenostylis stenocarpa), an underutilized West African food legume. One- and two-dimensional polyacrylamide gel electrophoresis was then used to analyze the albumin fraction, galactose-specific lectins purified on immobilized galactose-Sepharose 4B, and abundant non-lectin seed proteins left over following affinity chromatography. N-terminal sequencing of prominently resolved polypetide bands led to identification of proteins having sequence homology with characterized legume seed proteins, namely, mung bean seed albumin, pea alpha-fucosidase, soybean Kunitz-type trypsin inhibitor, an endochitinase, pea pathogenesis-related protein, and/or cowpea seed storage proteins. Minor lectin-like proteins lacking hemagglutinating activity against rabbit and human erythrocytes were also identified. Because proteins such as protease inhibitors, chitinases, pathogenesis-related proteins, and lectins are known to have antimetabolic effects, the findings from this study may have relevance in the acceptability, adoption, and utilization of AYB as human food.     

Characterization of phenolic profiles of Northern European berries by capillary electrophoresis and determination of their antioxidant activity

Berries are known to contain phenolic substances (i.e., flavonoids and phenolic acids), which comprise two large and heterogeneous groups of biologically active nonnutrients. This investigation evaluated the content and profile of the phenolic compounds present in six different berries found in Northern Europe. The latter included bilberry (Vaccinium myrtillus), cowberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccus), strawberry (Fragaria ananassa), black currant (Ribes nigrum), and red currant (Ribes rubrum). The study was focused on two areas. The first involved the extraction and analysis of berries for total phenolic content and determination of their antioxidant activity. The total phenolic level of berries was correlated with their antioxidant activity. Second, the berry extracts were analyzed by capillary electrophoresis to determine the content and profile of selected bioactive compounds. The analytes of interest included trans-resveratrol, cinnamic acid, ferulic acid, p-coumaric acid, quercetin, and morin.     

Differential recovery of lupin proteins from the gluten matrix in lupin-wheat bread as revealed by mass spectrometry and two-dimensional electrophoresis

Bread made from a mixture of wheat and lupin flour possesses a number of health benefits. The addition of lupin flour to wheat flour during breadmaking has major effects on bread properties. The present study investigated the lupin and wheat flour protein interactions during the breadmaking process including dough formation and baking by using proteomics research technologies including MS/MS to identify the proteins. Results revealed that qualitatively most proteins from both lupin and wheat flour remained unchanged after baking as per electrophoretic behavior, whereas some were incorporated into the bread gluten matrix and became unextractable. Most of the lupin α-conglutins could be readily extracted from the lupin-wheat bread even at low salt and nonreducing/nondenaturing extraction conditions. In contrast, most of the β-conglutins lost extractability, suggesting that they were trapped in the bread gluten matrix. The higher thermal stability of α-conglutins compared to β-conglutins is speculated to account for this difference.     

Proteomic analysis by two-dimensional gel electrophoresis and starch characterization of Triticum turgidum L. var. durum cultivars for pasta making

Criteria for durum wheat quality are continuously evolving in response to market pressure and consumer's preference. Specific attributes of durum wheat for different end products require more rapid and objective means to grade and classify wheat parcels based on processing potential. A total of 10 durum wheat cultivars were compared for compositional, protein, and starch characteristics. Mean values for the gross composition differed for total protein, gluten, and starch. Two-dimensional electrophoresis (2DE) analysis showed the proteome diversity among the cultivars. As shown by the principal component analysis (PCA) applied to 2DE data of gliadin and glutenin fractions, cultivars differed mainly from the number of proteins and levels of protein expression. As determined by the rapid viscoanalyzer (RVA), swelling power, starch damage, amylose content, and starch pasting properties of 10 cultivars differed significantly. 2DE fingerprinting and amylose content seemed to distinguish specific cultivars being useful tools for selecting suitable durum wheat cultivars for pasta making.     

Use of two-dimensional electrophoresis to evaluate proteolysis in salmon (Salmo salar) muscle as affected by a lactic fermentation

Two-dimensional electrophoresis was used to study proteolysis in salmon fillets inoculated or not with the starter culture Lactobacillus sake LAD. Protein fragments appeared increasingly with time in both samples, indicating that the main quantitative changes were due to endogenous enzymes. In the most acidic zone (pI = 4-6. 20) particularly, proteolysis was overall independent from processing. In contrast, fermentation had a significant effect in the pH range 6.20-8.35, suggesting a specificity of the bacterial proteases of L. sake toward alkaline to slightly acidic proteins. Furthermore, fragments surrounding tropomyosin (apparent pI = 4.70) appeared in fermented samples, indicating that the protein may be a suitable substrate for the metabolism of L. sake LAD.     

Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

Exploration of beer proteome using OFFGEL prefractionation in combination with two-dimensional gel electrophoresis with narrow pH range gradients

Two-dimensional gel electrophoresis in combination with mass spectrometry has already been applied successfully to study beer proteome. Due to the abundance of protein Z in beer samples, prefractionation techniques might help to improve beer proteome coverage. Proteins from four lager beers of different origins were separated by two-dimensional electrophoresis (2-DE) followed by tandem mass spectrometric analysis. Initially 52 proteins mostly from Hordeum vulgare (22 proteins) and Saccharomyces species (25 proteins) were identified. Preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2-DE to improve its resolution power. As a result of this combined approach, a total of 70 beer proteins from Hordeum vulgare (30 proteins), from Saccharomyces species (31 proteins), and from other sources (9 proteins) were identified. Of these, 37 proteins have not been previously reported in beer samples.     

转Bt-cry1Ab玉米花粉对意大利蜜蜂生长发育及体内酶活性的影响

自10年前首次种植转基因植物以来,转基因作物已在全球得到大面积推广。这些转基因作物中绝大多数品种具有抗虫基因能够增强作物对靶标昆虫的抗性。同时转抗虫基因植物可能带来的生态安全和环境问题也引起了国际社会广泛的关注。鉴于此,本实验开展了转Bt-cry1Ab基因抗虫玉米花粉     

The characterization of Nigerian varieties of pepper, Capsicum annuum and Capsicum frutescens by SDS- polyacrylamide gel electrophoresis of seed proteins

The possibility of using electrophoresis to characterize varieties of pepper, Capsicum annuum and Capsicum frutescens cultivated in Nigeria was investigated. The SDS- polyacrylamide gel electropherogram of extracted total seed proteins of 10 breeding lines in each of the 6 varieties investigated, revealed a pattern in which 12 polypeptide bands with apparent molecular weight range of 22 to 98 kilodaltons could be distinguished. The result showed that the six varieties could be characterized on the basis of presence/absence and staining intensities of 7 polypeptide bands. It is suggested that SDS- polyacrylamide gel electrophoresis of seed proteins provides a useful analytical technique for the characterization of varieties of pepper and there may be genotype duplicates in the collection of Nigerian Capsicum germplasm.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of gentically modified crops. 3. Assessing unintended effects

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.