Application of proteomics to hordein screening in the malting process

This study was undertaken to investigate the effect of the malting process on hordein composition. For this purpose, combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the method of isotopic peptides labeling iTRAQ was used. Barley proteins are essential components determining the quality of both malt and beer. Since hordeins represent the most abundant proteins accounting for about 40-50% of total protein fraction of mature barley grain, our research was focused on them. In this respect, the proteins of interest were extracted from milled samples of barley grain, germinated barley grain (samples collected at different time intervals), green malt and malt, respectively. Particular hordein extracts were firstly fractionated via SDS- PAGE, which was used as a relatively rapid and reliable technique providing information about hordein profile of analyzed samples. Then, separated proteins were in-gel digested and resulting peptides were measured by mass spectrometry. In addition, the chosen proteins, after in-gel digestion, were subjected to the iTRAQ method and the screening of proteins during malting process was evaluated. Our results have revealed that most of the hordein components present in the barley grain can be found in all stages of the malting process as well as in the final malt. The amount of hordeins decreases during the malting process; in the case of C hordein, the protein decrease is approximately 65%. On the other hand, significant degradation of D hordein was detected. The suggested procedure can be used to follow the development of the hordein profile during germination, which is of great technological importance in beer production.     

Rapid and highly sensitive protein extraction via cobalt oxide nanoparticle-based liquid-liquid microextraction coupled with MALDI mass spectrometry

A new approach for rapid and highly sensitive protein extraction using cobalt oxide nanoparticles modified with cetyltrimethylammonium (Co(3)O(4)/CTA(+) NP) using nanoparticle-based liquid-liquid microextraction (NP-LLME) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully demonstrated. For the first time, the metal oxide NPs (Co(3)O(4)/CTA(+) NP) prepared in the organic phase (toluene) have been successfully applied for the extraction and preconcentration of proteins from sample solutions and complex samples via electrostatic forces involved between the metal oxide NP and proteins. Lysozyme was used as the model protein to investigate the optimal extraction parameters of the current approach. The optimal conditions were obtained at pH > pI for 10 min of incubation time (extraction time) with 3% salt (NaCl) addition. The Co(3)O(4)/CTA(+) NP was successfully applied for the highly sensitive analysis of an array proteins such as insulin, chymotrypsinogen and lysozyme from aqueous solution, protein mixture and milk samples in nanoparticle-based liquid-phase microextraction coupled with MALDI-MS. The potentiality of the NP-LLME using Co(3)O(4)/CTA(+) NP for the extraction of proteins was also compared with other types of NP-liquid phase microextraction (LPME) methods. The current approach offers distinct advantages including rapidity, straightforwardness, high sensitivity for washing- and separation-free MALDI-MS analysis of proteins.     

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.     

Characterization of Xylanase and Cellulase Produced by a Newly Isolated <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> N2 and Its Efficient Saccharification of Barley Straw

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9 U/mL) and CMCase (5.61 U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65 °C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65 °C. The FPase was optimally active at pH 5.0 and 60 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.     

Separation and characterization of basic barley seed proteins

Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method.     

Studying ancient crop provenance: implications from δ(13)C and δ(15)N values of charred barley in a Middle Bronze Age silo at Ebla(NW Syria)

The discovery of a storeroom full of barley and other cereals (L.9512) in the proto-historic site of Ebla has provided a unique opportunity to study the centralized storage system of the early city-state from a different perspective. Epigraphic evidence available within the site reveals a complex system of taxation which included gathering grain tributes from satellite sites and redistributing semi-finished products such as flour. In this paper, we intend to explore the possibilities of a combined approach to studying the storage system, based on estimated barley grain volumes and δ(13)C-δ(15)N analyses. This approach is used to distinguish between grain from different harvesting sites and to identify any grain cultivated using special agricultural practices (e.g. manuring or irrigation). The basic assumption for this kind of analysis is that the growth-site conditions, natural or anthropogenic, of harvested cereals are reflected in their grain size and δ(13)C-δ(15)N values. Since the remains found in the storeroom were charred, the first task was to evaluate the effect of carbonization on the δ(13)C-δ(15)N and the size of the grains. Thus, the effect of charring was tested on modern samples of Syrian barley landraces. Once it had been ascertained that fresh grains reduced to charred remains retain their original biometric and isotopic traits, the ancient material was examined. Thirteen groups were identified, each characterized by a specific average volume and specific carbon and nitrogen values. The analysis revealed that what had first appeared to be a homogeneous concentration of grain was in fact an assemblage of barley harvested from different sites.     

Detection of protein coatings on nanoparticles surfaces by ToF‐SIMS and advanced electron microscopy

In this study, time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and advanced electron microscopy (scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM)) were applied to detect and analyse different nano‐scaled protein coatings on gold nanoparticles (NP). The NP were coated with collagen type I and fibronectin as well as different combinations of these proteins. These two main proteins in human cell organization and tissue formation were identified with the aid of ToF‐SIMS by typical amino acid mass peak detection. In addition, the protein‐coated particles were investigated by TEM and SEM to get information about the protein structure, the protein layer thickness on the particle surfaces and the reaction of NP in different protein solutions. In this study, a differentiation of diverse protein induced particle agglomeration was proven. The investigations of this study were part of the Specific Targeted Research Project CellNanoTox (project no. NMP4‐CT‐2006‐032731) funded by the European Commission under the 6th EU Framework Programme for Research and Technological Development. Copyright © 2013 John Wiley & Sons, Ltd.     

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods—trichloroacetic acid/acetone, Mg/NP‐40, and phenol/ammonium acetate—were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP‐40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.     

Identification of barley and rye varieties using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry with neural networks

Cereal varieties are normally identified using time-consuming methods such as visual examination of either the intact grain or one-dimensional electrophoretic patterns of the grain storage proteins. A fast method for identification of wheat (Triticum aestivum L.) varieties has previously been developed, which combines analysis of alcohol-soluble wheat proteins (gliadins) using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry with neural networks. Here we have applied the same method for the identification of both barley (Hordeum vulgare L.) and rye (Secale cereale L.) varieties. For barley, 95% of the mass spectra were correctly classified. This is an encouraging result, since in earlier experiments only a grouping into subsets of varieties was possible. However, the method was not useful in the classification of rye, due to the strong similarity between mass spectra of different varieties.     

Engineering the interface between glucose oxidase and nanoparticles

The behavior of glucose oxidase (GOx) on gold nanoparticles (NPs) was investigated as a function of (1) NP surface chemistry, (2) stabilizing protein additives, and (3) protein microenvironment. GOx secondary structure and unfolding was probed by circular dichroism (CD) spectroscopy and fluorescence, and GOx enzymatic activity was measured by a colorimetric assay. We also examined the activity and structure of GOx after displacement from the NP surface. Generally, GOx behavior was negatively impacted by conjugation to the NP, and conjugation conditions could vary the influence of the NP. Surface chemistry and protein microenvironment could improve behavior, but addition of stabilizing proteins negatively influenced activity. After displacement from the NPs, GOx tended to remain unfolded, indicating that the interactions with the NP were irreversible.     

Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress

Two spring wheat varieties Ningchun 4 and Chinese Spring with good and poor resistance to abiotic stress, respectively, were used to investigate proteomic changes in the developing grains under drought stress by a comparative proteomics approach. A total of 152 protein spots showed at least twofold differences in abundance on two-dimensional electrophoresis (2-DE) maps, of which 28 and 68 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry, respectively. Of the 96 identified protein spots, six different expression patterns were found and they were involved in stress/defense/detoxification, carbohydrate metabolism, photosynthesis, nitrogen metabolism, storage proteins and some other important functions. Comparative proteomic analysis revealed that under the drought conditions the decreased degree of ascorbate peroxidases was more significant in Chinese Spring than in Ningchun 4 during grain development whereas translationally controlled tumor protein, which was significantly upregulated at 14 DAF, was present in Ningchun 4 and absent in Chinese Spring. The Rubisco large subunit displayed an upregulated expression pattern in Ningchun 4. In addition, two drought-tolerant proteins, triosephosphate isomerase and oxygen-evolving complex showed B and F type expression patterns in Chinese Spring, but D and B types in Ningchun 4, respectively. These differentially expressed proteins might be responsible for the stronger drought resistance of Ningchun 4 compared to Chinese Spring.     

Polymeric Nanoparticles Amenable to Simultaneous Installation of Exterior Targeting and Interior Therapeutic Proteins

Effective delivery of therapeutic proteins is a formidable challenge. Herein, using a unique polymer family with a wide‐ranging set of cationic and hydrophobic features, we developed a novel nanoparticle (NP) platform capable of installing protein ligands on the particle surface and simultaneously carrying therapeutic proteins inside by a self‐assembly procedure. The loaded therapeutic proteins (e.g., insulin) within the NPs exhibited sustained and tunable release, while the surface‐coated protein ligands (e.g., transferrin) were demonstrated to alter the NP cellular behaviors. In vivo results revealed that the transferrin‐coated NPs can effectively be transported across the intestinal epithelium for oral insulin delivery, leading to a notable hypoglycemic response.     

Nanosized silver–palm pollen nanocomposite,green synthesis,characterization and antimicrobial activity

Palm pollen (PP) has been widely used in nutrition, pharmaceutical and cosmetic industries. In the present study, we explored the potential of PP in the synthesis of a silver nanoparticle (Ag NP). PP was used as both reducing and stabilizing agent. The Ag/PP nanocomposite was examined by field emission electron microscopy, X-ray diffraction, Fourier transform infrared (FT-IR) spectroscopy, ultraviolet spectroscopy and zeta potential measurement. The biosynthesized NPs showed surface plasmon resonance centered at 425 nm with an average particle size measured to be 23 nm and a zeta potential of ?30.9 mV. Prominent FT-IR signals were obtained and ascribed to phenolic and carbohydrate compounds involved in the formation of the Ag NPs, and proteins which participated in stabilization of the Ag NPs. The biologically synthesized Ag NPs were found to be extremely effective against E. coli (13.8 ± 0.25 mm) with a minimum inhibitory concentration of 20 µg/mL. Thus, such biosynthesized Ag NPs can be used in medicinal applications.     

Construction of Three Single‐Chain Antibody Fragment Variable Fusion Structures for Sensitive Detection of Nucleocapsid Protein of Hantaan Virus

Abstract

Three single‐chain fragment variable (scFv) fusion structures were constructed for use in rapid and sensitive detection of nucleocapsid protein (NP) of Hantaan virus. The detection of NPs on glass chips was signalized by enzyme labeling or fluorescence dye Cy3, or Cy5 cluster nanoparticles. The sensitivity of the methods with different signal systems was evaluated and compared. The detection limits of scFv‐alkaline phosphatase fusion, fluorescence labeling (scFv‐Cy3), and nanoparticles labeling (scFv‐SBP‐streptavidin‐nanoparticle) were 0.1 µg/mL, 1 ng/mL, and 0.1 ng/mL NP, respectively, which were all lower than that in a conventional enzyme‐linked immunosorbent assay (ELISA) (1 µg/mL). Twenty Hantaan virus isolates were detected using the proposed methods.     

Analysis of Fluorescent Proteins with a Nanoparticle Probe

This letter presents the first application of high energy, single nanoparticle probes (e.g., 520 keV Au(400) 2nm NP) in the characterization of surfaces containing fluorescent proteins (e.g., GFP variants) by their co-emitted photon, electron and secondary ion signals. NP induced protein luminescence increases with the NP incident energy, is originated by the NP impact and is transferred to the protein fluorophor via electronic energy transfer. Multi-electron emission is observed per single NP impacts and their distributions are specific to the target morphology and composition. Fragment ions of protein sub-units consisting of 2-7 amino acid peptides are observed under individual NP impacts that can be correlated to the random protein orientation relative to the impact site (e.g., outer layer or "skin" of the protein).     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.     

Electrochemical Study of the Lipid‐Transfer Protein

Electrochemical study of barley grain lipid‐transfer protein (LTP) revealed that it may belong to the metal‐binding protein class. Using differential pulse polarography the presence of Cu(II) and Zn(II) ions in the native LTP structure was proved, as well as its affinity for binding Ni(II) ion. Application of a more sensitive electroanalytical technique, such as anodic stripping voltammetry with analyte preconcentration, revealed the presence of Pb(II) and Cd(II) ions and also enabled the following Hg(II) ion binding. Possible biological role of LTP in plant stress response and its contribution to barley phytoextraction potential are discussed.     

Two-dimensional polyacrylamide gel electrophoresis, with immobilized pH gradients in the first dimension, of barley seed proteins: discrimination of cultivars with different malting grades.

The suitability of high-resolution two-dimensional gel electrophoresis for barley cultivar discrimination and for classification with respect to their malting properties was studied. Seed proteins of 14 barley cultivars with different malting qualities were extracted with urea/dithiothreitol/Nonidet P-40 buffer and subjected to two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT). The results of IPG-DALT were compared to the protein patterns obtained by a standard technique, sodium dodecyl sulfate polyacrylamide gel electrophoresis of hordeins. Sodium dodecyl sulfate-gel electrophoresis yielded seven different "B" and four different "C" hordein patterns; "A" and "D" hordein patterns were uniform in all cultivars tested. Four cultivars could be distinguished unequivocally, the others were classified into three groups containing between two and five cultivars. In contrast to these findings. IPG-DALT yielded three different "A", eight different "B", four different "C" and two different "D" hordein patterns. When the "A", "B", "C" and "D" hordein patterns were combined, ten cultivars exhibited unique hordein patterns whereas the remaining ones were classified into two groups containing two cultivars each. Moreover, when albumin and globulin proteins were used for evaluation in addition to the hordeins, all cultivars could be discriminated by IPG-DALT. IPG-DALT, performed on small-scale and/or ready-made gels, proved to be an ideal complementary system to one-dimensional electrophoretic methods for routine seed testing purposes because of its speed, reliability, and simplicity. IPG-DALT was also applied to study the relationship between the different polypeptide patterns and the malting quality. Although cultivars with identical one-dimensional protein patterns but different malting quality could be successfully differentiated by IPG-DALT, a direct correlation between specific protein spots or protein patterns to the malting quality was not found within the cultivars tested.