Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Diversity of Novel Glutenin Subunits in Bread Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on two-dimensional gel electrophoresis with isoelectric focusing (wide range of pH 3–10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current study.The proteins were extracted by Trichloracetic acid (TCA) method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension.The spots of proteins were visualized by staining with Coomassie Brilliant Blue.After analysis with software (ImageMaster 2D),122 different proteins were detected;isoelectric point (pI),Molecular weight (MW) and relativevolume of each protein in the pollen were also discovered.This is the first high-resolution,two-dimensional protein map of the pollen ofHumulus scandens Lour in China.Our finding has built a solid foundation for identification,characterization,gene cloning and standardization of allergenic proteins in the pollen ofHumulus scandens Lour for further studies.     

Genetic variation in wheat endosperm proteins: an analysis by two-dimensional electrophoresis using intervarietal chromosomal substitution lines

Summary The major endosperm proteins in a range of genotypes of hexaploid wheat have been fractionated by two-dimensional electrophoresis. The genotypes included nine varieties and forty four intervarietal substitution lines in which chromosomes 1A, 1B, 1D, 6A, 6B or 6D from eight of the varieties have been introduced one at a time into a common genetic background. The appearance of different protein subunits was often correlated with a chromosome substitution. This showed that many of the genes for the high molecular weight protein subunits (molecular weight range 55,000 to 140,000 determined by SDS polyacrylamide gel electrophoresis) are specified by chromosomes 1A, 1B and 1D while many of the lower molecular weight subunits (molecular weight range 30,000 to 45,000) are specified by chromosomes 6A, 6B and 6D. The different protein subunits correlated with chromosome substitution could not always be recognised in the varietal source of the substituted chromosome. The different subunits specified by homologous chromosomes in different wheat varieties may differ in isoelectric point and/or molecular weight.     

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.     

A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis

Proteins extracted from embryos, endosperms and leaves of rice were separated by two-dimensional electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 electroblotted proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino-acid sequences of 27 out of 85 proteins were determined in this manner. The N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Among proteins, 11 could be sequenced after deblocking by in situ treatment with pyroglutamyl peptidase. The internal amino-acid sequences of 23 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. The concanavalin A-peroxidase method was used to determine whether the 85 proteins were glycosylated and the diagonal electrophoresis method was used to determine whether they contained disulphide bonding. Finally, we constructed a data-file of rice proteins including information on relative molecular weight, isoelectric point, amino-acid sequence, sequence homology, glycosylation, and the presence of disulphide bonding.     

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.     

Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis

To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.     

Immunodetection and immunolocalization of tryptophanins in oat (Avena sativa L.) seeds

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat (Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat (Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley (Hordeum vulgare L.) seed extracts but not in rice (Oryza sativa L.), maize (Zea mays L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and lentil (Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.     

Microheterogeneity of Micro tubule-Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtubule-associated tau proteins using tau-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to tau proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, with apparent molecular weights between 56,000 and 66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that greater than 30 isoforms of tau exist. The tau proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of tau is not induced by cycling of microtubules, because two-dimensional immunoblots of tau from total brain are almost identical to those of tau from cycled tubules. Adult rat brain tau, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does tau from 7-day-old rats, which appears as only one band on SDS gels. After dephosphorylation of cow brain tau with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of tau variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with tau labeled in vivo with 32PO4 also indicate that the more acidic tau proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of tau proteins exists at all ages and is due, at least in part to differences in the state of phosphorylation of tau isoforms.     

Mapping and proteomic analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum aestivum L.)

Albumins and globulins of wheat endosperm represent 20% of total kernel protein. They are soluble proteins, mainly enzymes and proteins involved in cell functions. Two-dimensional gel immobiline electrophoresis (2DE) (pH 4-7) × SDS-Page revealed around 2,250 spots. Ninety percent of the spots were common between the very distantly related cultivars ‘Opata 85’ and ‘Synthetic W7984’, the two parents of the International Triticeae Mapping Initiative (ITMI) progeny. ‘Opata’ had 130 specific spots while ‘Synthetic’ had 96. 2DE and image analysis of the soluble proteins present in 112 recombinant inbred lines of the F9-mapped ITMI progeny enabled 120 unbiased segregating spots to be mapped on 21 wheat (Triticum aestivum L. em. Thell) chromosomes. After trypsic digestion, mapped spots were subjected to MALDI-Tof or tandem mass spectrometry for protein identification by database mining. Among the ‘Opata’ and ‘Synthetic’ spots identified, many enzymes have already been mapped in the barley and rice genomes. Multigene families of Heat Shock Proteins, beta-amylases, UDP-glucose pyrophosphorylases, peroxydases and thioredoxins were successfully identified. Although other proteins remain to be identified, some differences were found in the number of segregating proteins involved in response to stress: 11 proteins found in the modern selected cultivar ‘Opata 85’ as compared to 4 in the new hexaploid `Synthetic W7984’. In addition, ‘Opata’ and ‘Synthetic’ differed in the number of proteins involved in protein folding (2 and 10, respectively). The usefulness of the mapped enzymes for future research on seed composition and characteristics is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of cytoplasmic ribosomal proteins of gateway barley and its mutant

The proteins of the cytoplasmic ribosomes isolated from dry embryos of Gateway barley and its virescens mutant were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The monosomes of both the lines gave similar patterns with 60 basic proteins. Upon dissociation of the monosomes, for the mutant, the basic proteins of the large subunits migrated more slowly than those of the normal and lacked three proteins but had three additional spots. Also, the proteins of the small subunits differed. The mutant lacked three of the proteins present in the normal but had three additional spots. Therefore, the large and small subunits contained a total of 34 and 41 basic proteins, respectively, in both the lines. There were several spots with identical electrophoretic mobilities in the small and large subunits of these two lines.     

A Double-Isotope Procedure for Examining Protein Microheterogeneity: Multiple Forms of Fast-Transported Glycoproteins and Sulfoproteins Possess a Common Polypeptide Chain

Several fast-transported proteins that appear as single bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolve into multiple spots during isoelectric focusing. A method was devised for determining if such microheterogeneity in net charge indicates that individual polypeptides have been posttranslationally modified to differing extents. Dorsal root ganglia were pulse-labeled with [35S]methionine and either [3H]leucine or [3H]proline, proteins fast-transported into peripheral sensory axons were separated by two-dimensional gel electrophoresis, and isotope incorporation ratios of proteins associated with individual gel spots were determined. When four microheterogeneous glycoproteins were analyzed, each protein "family" showed markedly similar isotope ratios for its three to seven characteristic spots. Such ratios differed between families by almost twofold. In addition, a group of nonglycosylated, sulfate-containing proteins was identified as a family on the basis of the similar isotope incorporation ratios of its component spots. These results suggest that protein microheterogeneity can result from variable sulfation of tyrosine residues as well as from variation in sialic acid-containing oligosaccharide side-chains. More generally, the method can be utilized to test for protein microheterogeneity in cases where the amounts of protein are too low to permit peptide mapping analysis and where the nature of the charge-altering modification is unknown.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Electrophoretic and Immunological Properties of Folate-binding Protein Isolated from Bovine Milk

Changes of the folate-binding protein (FBP) concentration in bovine milk after parturition were investigated. The FBP was highly purified from mature milk by affinity chromatography. The purified FBP showed a single protein band in polyacrylamide gel electrophoresis and was immunologically homogenous in double immunodiffusion. However, in two-dimensional gel electrophoresis, the FBP was separated into several spots in isoelectric focusing in the first dimension, and each spot also showed two molecular weights in SDS-gel electrophoresis in the second dimension. But these FBP molecules were immunologically identical with each other. The neuraminidase treatment obviously diminished the number of isoelectric points of the FBP. Thus, the variety of FBP molecules was at least partially due to the variability of the sialic acid content in the carbohydrate moieties. Moreover, the milk FBP showed species-specificity among mammals immunologically as well as physicochemically.     

Identification and molecular characterisation of hordoindolines from barley grain

Grain texture in barley is an important quality character as soft-textured cultivars have better malting quality. In wheat, texture is considered to be determined by the puroindolines, a group of basic hydrophobic proteins present on the surface of the starch granule. Hard wheats have been proposed to lack puroindoline a or to have mutant forms of puroindoline b which do not bind to the granule surface. Analysis of six barley cultivars (three soft-textured and three hard) showed that all contained proteins homologous to wheat puroindoline b, but PCR analysis failed to show any differences in amino acid sequences similar to those which have been proposed to determine textural differences in wheat. Southern blot analysis showed two hordoindoline b genes which were isolated and shown to encode proteins with 94% sequence identity. Expression of hordoindoline b mRNA occurred in the starchy endosperm and aleurone layer of the developing seed, but not in the embryo. Analysis of seven soft- and six hard-textured barley varieties showed that all contained hordoindoline a except two hard varieties (Sundance, Hart) which were subsequently shown to both lack hordoindoline a mRNA. It was therefore concluded that there is not a clear relationship between the presence of hordoindoline a and grain texture in barley.     

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.     

Proteomic analysis of aneuploid lines in the homeologous group 1 of the hexaploid wheat cultivar Courtot

Three monosomic lines (MSLs) and three nullisomic lines (NSLs) of the homeologous group 1 and one euploid line of the bread wheat Triticum aestivum cultivar Courtot were used in a proteomic approach to investigate the effects of zero, one or two doses of chromosomes 1A, 1B and 1D on the amount of endosperm proteins. Polypeptides whose amounts changed significantly between each aneuploid line and the euploid line were identified using image analyses of two-dimensional gel electrophoresis patterns resulting from specific endosperm protein extractions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry were also used for protein identification. Removing one chromosome or a chromosome pair allowed varying responses to be observed for the remaining endosperm protein genes. Compensation phenomena for the high molecular weight glutenin subunits (HMW-GS) were detected only in the MSLs. Subunits Bx7, By8 and Dy12 were the only HMW-GS overexpressed (from 152-737%) when chromosomes 1A or 1B or 1D were at hemizygous state. Thirteen new protein spots were detected only in the NSL1D, and seven were identified as HMW-GS analogs. These seven new spots may result from the expression of inactive genes. The HMW-GS were of significantly higher volume in MSLs, whereas the low molecular weight glutenin subunits and the gamma-gliadins were of lower volume in aneuploid lines. Most of the down-regulated proteins in the MSLs were storage proteins encoded at loci located on another chromosome pair. Complex regulations between chromosomes and loci of the homeologous groups 1 and 6 in bread wheat are discussed.