Comparative 2-DE protein analysis in a 3-D geometry gel

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.     

Interference by Tris buffer in the estimation of protein by the Lowry procedure

Tris buffers were found to distort the measurement of protein by the Lowry method both by decreasing chromophore development with protein and by contributing blank color. Tris at an assay concentration of 0.37 mm markedly affects measured results. Similar Tris effects were observed at all wavelengths between 450 and 800 nm and with diverse protein samples. The distortion due to Tris is not correctable by simple blank correction, but it can be overcome by incorporating the same amount of Tris in the standards used. The distortion at Tris concentrations <0.15 mm appears to be within tolerable limits. No interference or distortion was observed with sodium phosphate buffer to an assay concentration of 40 mm. An automated Lowry procedure is also presented which gives excellent correlation with the manual method and an average coefficient of variation of <4%.     

Protein spot detection and quantification in 2-DE gel images using machine-learning methods

Two-dimensional gel electrophoresis (2-DE) is the most established protein separation method used in expression proteomics. Despite the existence of sophisticated software tools, 2-DE gel image analysis still remains a serious bottleneck. The low accuracies of commercial software packages and the extensive manual calibration that they often require for acceptable results show that we are far from achieving the goal of a fully automated and reliable, high-throughput gel processing system. We present a novel spot detection and quantification methodology which draws heavily from unsupervised machine-learning methods. Using the proposed hierarchical machine learning-based segmentation methodology reduces both the number of faint spots missed (improves sensitivity) and the number of extraneous spots introduced (improves precision). The detection and quantification performance has been thoroughly evaluated and is shown to compare favorably (higher F-measure) to a commercially available software package (PDQuest). The whole image analysis pipeline that we have developed is fully automated and can be used for high-throughput proteomics analysis since it does not require any manual intervention for recalibration every time a new 2-DE gel image is to be analyzed. Furthermore, it can be easily parallelized for high performance and also applied without any modification to prealigned group average gels.     

A high pH discontinuous buffer system for resolution of isozymes in starch gel electrophoresis

A Tris-citrate pH 9.5 gel/borate pH 8.2 electrode discontinuous buffer system for starch gel electrophoresis of proteins was developed to resolve iso- and allozymes of aspartate aminotransferase in frogs (Hyla crucifer). This buffer system also enhanced resolution of NADP-dependent malate dehydrogenase and the L-lactate dehydrogenase-A locus in this species. It provided good resolution of NAD-dependent malate dehydrogenase in esocid fishes, and esterases, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, and S-aconitate hydratase in ambystomatid salamanders. Variation suppressed by other buffers was revealed by this buffer for some enzyme encoding loci, while at other loci, this buffer suppressed electromorph variability. The concentration of tris(hydroxymethyl)aminomethane in gels made with this buffer was much higher than in pH 8.7 "Poulik" gels, but running characteristics of the two gel types were similar. Gels made with this new buffer were less prone to splitting and "warping" than Poulik gels, and were easier to handle. When screening a given taxon for enzyme variability, tests using multiple buffers are essential to maximize the amount of electrophoretically detectable variation.     

Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca²⁺ -dependent protein kinase. The Ca²⁺ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca²⁺ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca²⁺ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca²⁺ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca²⁺ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K_m values for ATP, and in Ca²⁺ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca²⁺ -dependent protein kinases present in the soluble phase.     

Toward a high resolution 2-DE profile of the normal human liver proteome using ultra-zoom gels

The human liver is the largest organ in the body and has many important physiological functions. A global analysis of human liver proteins is essential for a better understanding of the molecular basis of the normal functions of the liver and of its diseases. As part of the Human Liver Proteome Project (HLPP), the goal of the present study was to visualize and detect as many proteins as possible in normal human livers using two-dimensional gel electrophoresis (2-DE). We have constructed a reference map of the proteins of human normal liver that can be used for the comprehensive analysis of the human liver proteome and other related research. To improve the resolution and enhance the detection of low abundance proteins, we developed and optimized narrow pH range ultra-zoom 2-DE gels. High resolution patterns of human liver in pH gradients 4.5–5.5, 5–6, 5.5–6.7, 6–9 and 6–11 are presented. To improve the poor resolution in the alkaline pH range of 2-DE gels, we optimized the isoelectric focusing protocol by including sample application using cup loading at the anode and incorporating 1.2% hydroxyethyl disulfide, 15% 2-propanol and 5% glycerol in the rehydration buffer. Using the optimized protocol, we obtained reproducibly better resolution in both analytical and preparative 2-DE gels. Compared with the 2386 and 1878 protein spots resolved in the wide range 3–10 and 4–7 pH gradients respectively, we obtained 5481 protein spots from the multiple (overlapping) narrow pH range ultra-zoom gels in the range of pH 4.5–9. The visualized reference map of normal human liver proteins presented in this paper will be valuable for comparative proteomic research of the liver proteome.     

Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C₃ plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO₂ concentrations varying from 1 to 20% in air. When they were transferred back into CO₂-free air, CO₂ was rapidly released. It originated from dissolved CO₂ and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO₂ from bicarbonate. The data were fitted to a model which accounts for the CO₂/bicarbonate equilibrium in buffers with different CO₂ concentrations and initial pH values. From this, pH values and CO₂-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO₂ concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO₂ between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H⁺ per pH unit. The work shows that measurements of solubilized CO₂ are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl chlorophyll This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice.     

Evaluation of 2-DE protein patterns from pre- and postnatal stages of the mouse brain

Brains of the mouse from three developmental stages, embryo day 16 (Ed16), postnatal stage one week (1W) and eight weeks (8W), were distributed to different laboratories for a collaborative proteome analysis (The Human Brain Proteome Project). As one of the laboratories involved in this project, we separated total protein extracts of the brains by large gel 2-DE. From the 2-DE protein patterns a section was evaluated for each of the three stages according to resolution, reproducibility and quantitative changes using an image analysis software. The evaluated pattern section was selected to allow comparisons of 2-DE patterns between different laboratories on the basis of optimum separation. Changes in protein expression were analysed within two phases of development: Stage Ed16 versus stage 1W and stage 1W versus stage 8W. Out of the 200 protein spots evaluated 5-6% showed quantitative changes in the range of > or = 30% between two stages. The relationship in the frequency of up- and down-regulated protein spots differed between the two investigated phases. Most of the protein spots which showed altered expression between two stages were identified by MS. High quality in protein separation and evaluation is demonstrated.     

Evaluation of protein N-glycosylation in 2-DE: Erythropoietin as a study case

The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-alpha were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity ( approximately 25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetra-sialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.     

Analysis of protein changes during grape berry ripening by 2-DE and MALDI-TOF

Grape berry, a nonclimacteric fruit, during ripening turns from green, hard and acidic to coloured, soft and sweet. Many studies have focused on dynamic changes of mRNA levels, metabolites, sugars or individual proteins, but this is the first report of a proteomic approach applied to the screening of the most prominent variations that take place during berry ripening. Vitis vinifera cv. 'Nebbiolo Lampia' berries were collected at 10-day intervals, starting 1 month after flowering to complete ripe stage; total protein extracts from deseeded berries were separated by 2-DE. A total of 730 spots were detected in the 2-DE gels. 118 protein spots, differentially expressed during berry development, were subjected to MALDI-TOF analysis. Ninety-three of them were identified, corresponding to 101 proteins. The majority of proteins were linked to metabolism, energy and protein synthesis and fate. In comparison to published surveys of major berry proteins, fewer proteins related to stress response and more proteins related to cell structure were differentially expressed. Our data confirm a general decrease of glycolysis during ripening, and an increase of PR proteins in the range of 20-35 kDa. They furthermore suggest that oxidative stress decreases during ripening while extensive cytoskeleton rearrangement takes place in this period.     

Improvement in growth and yield attributes of cluster bean through optimization of sowing time and plant spacing under climate change scenario

Cluster bean (Cyamopsis tetragonoloba L.) yield has plateaued due to reduction in rainfall and rise in temperature. Therefore, its production cycle could not get appropriate water and temperature. It becomes important to standardize the sowing time and plant spacing of cluster beans in changing climate scenarios to get higher productivity. Therefore, a field study was conducted in 2019 at the Research area of MNS-University of Agriculture, Multan, Pakistan to evaluate the effect of four sowing times (15th May, 1st June, 15th June, and 1st July) and three plant spacings (10, 12 and 15 cm) on crop growth, yield, and physiological functions of cluster bean genotype BR-2017 under split plot arrangement under randomized complete block design (RCBD) with three replications. The sowing times (15th May, 1st June, 15th June, and 1st July) were placed in the main plot, while plant spacing (10, 12 and 15 cm) was maintained in subplots. The significant effect of sowing time and plant spacing was observed on pod plant⁻¹, pod length, grain yield, and 1000-grain weight. Results showed that 1st June sowing performed better over 15th May, 15th June, and 1st July, while plant spacing 15 cm about in all sowing times showed higher results on growth and yield parameters of cluster bean over plant spacing 10, 12, and 15 cm. The 1st June sowing time at 15 cm plant spacing showed 8.0, 22.7, and 28.5% higher grains pod^-1 than 15th May, 15th June, and 1st July sowing, respectively. Maximum grain yield was observed on 1st June in all three spacings (10, 12, and 15 cm). The chord diagram indicates that the crop has received optimum environmental conditions when sown 1st June over other sowing times. In conclusion, 1st June sowing with 15 cm plant spacing could be a good option to achieve maximum productivity of cluster bean under changing climate scenario.     

High performance concentration and gel filtration of rat urinary protein allergens

Allergies to laboratory animals, notably rats, have become an increasingly recognized occupational problem. Identification and isolation of the individual proteins causing allergic reactions, could form the basis for early recognition of sensitivity, diagnosis, control of degree of pollution of the environment and desensibilization treatments. Frequently, allergens originate from dried rat urine. Because earlier published methods were found unsatisfactory we have developed a new strategy for isolation of rat urinary proteins including a high performance technique for their mild concentration on hydroxyapatite. The concentrated allergens have been fractionated according to molecular size by high performace gel filtration and according to carbohydrate content by wheat germ lectin-Sepharose 6 MB affinity chromatography. The obtained fractions have been examined by denaturing and non-denaturing polyacrylamide gradient gel electrophoresis followed by sensitive staining procedures, and tested with respect to allergenicity by skin tests on allergic patients.     

Optimum concentration and pH of phosphate buffer for assay of cytochrome c oxidase in intact plant mitochondria

For measurement of cytochrome c oxidase activity in intact plant mitochondria the optimum concentration of K-Pi buffer and pH in the reaction was found to be 75 mM and 7.4 respectively. The suitable concentration of K-Pi buffer for suspending and storing mitochondria, however, was found to be 20 mM or lower. These requirements applied equally well for mitochondria from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), maize (Zea mays L.), and snap bean (Phaseolus vulgaris L.).     

Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS

Vibrio cholerae can be differentiated into epidemic and non-epidemic strains by sorbitol fermentation speed, but little research has been done on its mechanisms. In this study, we investigated differential protein expression of the two strains in response to sorbitol metabolism. V. cholerae strains were cultured in media with and without sorbitol, respectively. Proteins were separated by 2-DE, and those that showed different expression in the two media were identified by MALDI-TOF MS. Fifteen proteins in epidemic strains and 11 proteins in non-epidemic strains showed a different expression in sorbitol medium. Among them, 4 proteins were common to epidemic and non-epidemic strains. Gene sequence analysis showed that some mutations occurred in these proteins between the two strains. Potential functions of these proteins included sugar uptake, amino acid uptake, electron transport, sulfate and thiosulfate transport.     

Bromophenol blue protein assay: Improvement in buffer tolerance and adaptation for the measurement of proteolytic activity

A modification of the bromophenol blue dye binding procedure of protein estimation is described. Substritution of glycine/phosphoric acid, pH 2.6, for dilute acetic acid in the colour reagent extended the applicability of the procedure to protein solutions containing buffers of various pH values. This was, however, accompained by approximately 25% loss in the sensitivity of the procedure. The mofified reagent exhibited very marked tolerance to detergents and could be successfuly adapted for the measurement of proteolytic activity in acidic, neutral or alkaline pH ranges.     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.     

Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique

The Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) pilot studies have generated over 200 2-D gels from eight participating laboratories. This data includes 67 single-channel and 60 DIGE gels comparing 30 whole frozen C57/BL6 female mouse brains, ten each at embryonic day 16, postnatal day 7 (juvenile) and postnatal day 54-56 (adult); and ten single-channel and three DIGE gels comparing human epilepsy surgery of the temporal front lobe with a corresponding post-mortem specimen. The samples were generated centrally and distributed to the participating laboratories, but otherwise no restrictions were placed on sample preparation, running and staining protocols, nor on the 2-D gel analysis packages used. Spots were characterised by MS and the annotated gel images published on a ProteinScape web server. In order to examine the resultant differential expression and protein identifications, we have reprocessed a large subset of the gels using the newly developed RAIN (Robust Automated Image Normalisation) 2-D gel matching algorithm. Traditional approaches use symbolic representation of spots at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in spot modelling and matching. With RAIN, image intensity distributions, rather than selected features, are used, where smooth geometric deformation and expression bias are modelled using multi-resolution image registration and bias-field correction. The method includes a new approach of volume-invariant warping which ensures the volume of protein expression under transformation is preserved. An image-based statistical expression analysis phase is then proposed, where small insignificant expression changes over one gel pair can be revealed when reinforced by the same consistent changes in others. Results of the proposed method as applied to the HUPO BPP data show significant intra-laboratory improvements in matching accuracy over a previous state-of-the-art technique, Multi-resolution Image Registration (MIR), and the commercial Progenesis PG240 package.