Identification of wheat varieties using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and an artificial neural network

A novel tool for variety identification of wheat (Triticum aestivum L.) has been developed: an artificial neural network (ANN) is used to classify the gliadin fraction analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The robustness of this novel method with respect to various experimental parameters has been tested. The results can be summarised: (i) With this approach 97% of the wheat varieties can be classified correctly with a corresponding correlation coefficient of 1.0, (ii) The method is fast since the time of extracting gliadins from flour can be reduced to 20 min without significant decrease in overall performance, (iii) The storage of flour or extracts under standard conditions does not influence the classification ability (i. e. the generalisation ability) of the method, and (iv) The classification obtained is not influenced by the identity of the operator making the analysis. This study demonstrates that a combination of an ANN and MALDI-TOFMS analysis of the gliadin fraction provides a fast and reliable tool for the variety identification of wheat. Copyright 1999 John Wiley & Sons, Ltd.     

Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.     

The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I] concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combination of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.     

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.     

Two-dimensional electrophoresis and mass spectrometry identification of proteins bound by a murine monoclonal anti-cardiolipin antibody: a powerful technique to characterize the cross-reactivity of a single autoantibody

Antigenic cross-reactivity, i.e., the capacity of a single antibody to react with apparently dissimilar structures, is a common characteristic of autoantibodies produced during systemic lupus erythematosus (SLE), an autoimmune disease developed by humans and certain strains of mice. Characterization of the extent of cross-reactivity of SLE-related autoantibodies may help identify the immunogenic stimulus, or stimuli, of autoantibody-secreting B-lymphocytes. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was combined with mass spectrometry (MS) to identify cell proteins recognized by a single monoclonal autoantibody (mAb 4B7), derived from an (NZW x BXSB)F1 mouse and selected based on its capacity to react with cardiolipin, that binds to elements in the cytoplasm and nucleoli of HEp-2 cells as assessed by indirect immunofluorescence assay. Proteins from HL-60 extract were separated by 1-D and 2-D PAGE. Western blotting with mAb 4B7 after SDS-PAGE revealed four bands, two intensely labeled at 35 and 32 kDa, and two weaker ones at 20 and 60 kDa; three spots were detected after 2-D PAGE. After trypsin in-gel digestion of the three protein spots, MS yielded representative matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) Reflector or quadrupole-time of flight (Q-TOF) spectra. The three corresponding proteins were identified as the nucleolar phosphoprotein B23 (nucleophosmin), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and the 60 kDa Ro/SS-A RNP. Thus, these results showed that 2-D PAGE combined with MS constitutes a sensitive and powerful technique to characterize the full extent of cross-reactivity of a single mAb and may constitute a new approach to further characterize the immunogenic cellular components involved in the breakage of B-cell tolerance observed in SLE.     

Full-fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3-D analytical technique: Identification of new heterodimeric aggregation states

Wheat proteinaceous alpha-amylase inhibitors (alpha-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive alpha-AIs that share sequence homology, but differ in their specificity against alpha-amylases from different species and for their aggregation states. Wheat alpha-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive alpha-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of alpha-AIs (resistant to T > 70 degrees C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the alpha-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric alpha-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric alpha-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat alpha-AIs.     

The application of two-dimensional polyacrylamide gel electrophoresis to medical microbiology: molecular epidemiology of viruses and bacteria

A variety of molecular methods can be used to identify protein and nucleic acid markers with which to investigate the epidemiology of viruses and bacteria. This paper reviews the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for studying microbial molecular epidemiology. A small format 2-D PAGE system is described for locating protein markers in group B coxsackie viruses (CVB) and Haemophilus influenzae isolates. Representative isolates of CVB serotypes 2, 4, and 5 were compared by analysing the intracellular proteins present in CVB-infected HEp-2 cells by 2-D PAGE protein gels. Although some of the virus-induced proteins had similar electrophoretic mobilities, the three serotypes could be distinguished from each other on the basis of a major virus-induced protein of molecular weight between 39,000 and 43,000. Protein differences were demonstrated among six serotype 2 CVB (CVB-2) isolates. Four clinical CVB-2 isolates collected over a period of four months had indistinguishable two-dimensional protein profiles. Comparison of the two-dimensional protein profiles of cloned virus stocks prepared from a single clinical CVB isolate demonstrated that it was a heterogeneous virus population. The proteins of nontypable and type-b H. influenzae isolates were compared. Up to 160 proteins, detected by staining with Coomassie Brilliant Blue R, were resolved by 2-D PAGE. Although protein differences between individual bacterial isolates were detected, comparable two-dimensional protein profiles were found for the two groups of H. influenzae isolates. There was no similarity in the two-dimensional protein profiles of H. influenzae and Aeromonas. Potential protein markers were identified that may be useful in long-term studies of H. influenzae epidemiology.     

Explorative data analysis of two-dimensional electrophoresis gels

Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots on the gels for further investigation. The explorative approach in analysis of 2-D gels makes it possible, in a fast and convenient way, to screen many gels in order to determine the protein patterns that form clusters and could be selected for further examination.     

Maize pollen enzymes after two-dimensional polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate

Twelve enzymes from mature pollen grains of maize were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2-D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic-oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1-D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2-D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed.     

A procedure for the extraction and high resolution two-dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei.

Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.     

Identification of rat urinary glycoproteome captured by three lectins using gel and LC-based proteomics

Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.     

Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.     

Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.     

Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.     

Alternative sample preparation prior to two-dimensional electrophoresis protein analysis on solid lipid nanoparticles

The proteins adsorbing onto the surface of intravenously injected drug carriers are regarded as a key factor determining the organ distribution. Depending on the particle surface properties, certain proteins will be preferentially adsorbed, leading to the adherence of the particle to cells with the appropriate receptor. Therefore, the knowledge of the protein adsorption pattern and the correlation to in vivo behavior opens the perspective for the development of intravenous colloidal carriers for drug targeting. After incubation in plasma, the adsorbed proteins were analyzed using two-dimensional polyacrylamide gel electrophoresesis (2-D PAGE, 2-DE). The purpose of the present study was to develop an alternative separation method to separate solid lipid nanoparticles (SLN) carriers from plasma by gel filtration prior to 2-D PAGE. Via the specific absorption coefficients and a two-equation system, elution fractions were identified being practically plasma-free. This allows protein analysis on SLN which are typically in density too close to the density value of water to be separated by the standard centrifugation method. The SLN used for establishing the gel filtration were prepared in a way that they had a sufficiently low density to be additionally separated by centrifugation. The adsorption patterns obtained after separation with both methods were qualitatively and quantitatively identical, showing the suitability of the gel filtration.     

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.     

Phenolic compounds and the antioxidant activity of the bran,flour and whole grain of different wheat varieties

Total phenolic content and DPPH radical scavenging capability of the bran layer, flour made from endosperm and whole grain of wheat were determined. Fifteen different wheat samples of ten spring and five winter wheat varieties were analyzed. The spring wheat varieties were grown in both conventional and organic conditions. The total phenolic content of the bran layer found to be the highest (1258-3157 μg/g), followed by that of grains (168 - 459 μg/g) and the lowest of flour (44 - 140 μg/g). The bound phenolic acids were quantified by CE-DAD analysis after alkaline hydrolysis. Ferulic acid was a major compound among phenolic acids found in wheat varieties.