Dialysis-assisted two-dimensional gel electrophoresis

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

High resolution two-dimensional polyacrylamide gel electrophoresis

The combination of two different gel electrophoretic techniques in a two-dimensional separation procedure provides the resolution capacity required for the simultaneous separation and analysis of complex protein mixtures. This technique is therefore a powerful tool for the study of phenotypic expression.     

Virtual two-dimensional gel electrophoresis of high-density lipoproteins

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2-D gel analyses, virtual 2-D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post-translational modifications. Furthermore, its general ability to deliver ions from sub-picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top-down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2-D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.     

Software-induced variance in two-dimensional gel electrophoresis image analysis

Experimental variability in 2-DE is well documented, but little attention has been paid to variability arising from postexperimental quantitative analyses using various 2-DE software packages. The performance of two 2-DE analysis software programs, Phoretix 2D Expression v2004 (Expression) and PDQuest 7.2 (PDQuest), was evaluated in this study. All available background subtraction and smoothing algorithms were tested using both data generated from one single 2-DE gel image, thus excluding experimental variance, and with authentic sets of replicate gels (n = 5). A slight shift of the image boundaries (the "cropping area") caused both programs to induce variance in protein spot quantification of otherwise identical gel images. The resulting variance for PDQuest (CV(mean) = 8%) was approximately twice that for Expression (CV(mean) = 4%). In authentic sets of replicate 2-DE gels (n = 5), the experimental variance confounded the software-induced variance to some extent. However, Expression still outperformed PDQuest, which exhibited software-induced variance as high as 25% of the total observed variance. Surprisingly, the complete omission of background subtraction algorithms resulted in the least amount of software-based variance. These data indicate that 2-DE gel analysis software constitutes a significant source of the variance observed in quantitative proteomics, and that the use of background subtraction algorithms can further increase the variance.     

Spot volume vs. amount of protein loaded onto a gel: a detailed,statistical comparison of two gel electrophoresis systems

The long-term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and PDQuest 2-D analysis software, a reproducible 2-DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single-concentration gel (190 x 205 x 1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834-1846) the spatial and quantitative reproducibility of the two second-dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2-D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.     

Integrated polymer chip for two-dimensional capillary gel electrophoresis

Two-dimensional (2D) gel electrophoresis (GE) is one of the most powerful methods for nucleic acid and protein separation, but generally suffers from high laboratory efforts connected with high analysis costs. Therefore, we herein present the development of a miniaturized 2D capillary GE (CGE) device which allows for an efficient protein separation in analysis times of about 1.5 h. This integrated 2D-CGE chip comprises a first channel for isoelectric focussing (IEF), a second specially designed transfer channel, 300 parallel micro channels, each having a cross section of 50 microm x 50 microm, and buffer reservoirs. The present work discusses fabrication aspects, in particular the combination of different microfabrication technologies, experimental separation performances of isoelectric focussing (IEF) and CGE, and presents computer simulations and first experimental results of protein transfer from the first to the second dimension.     

Use of Legendre moments for the fast comparison of two-dimensional polyacrylamide gel electrophoresis maps images

In this paper, Legendre moments are calculated to extract the global information from a set of two-dimensional polyacrylamide gel electrophoresis map images. The dataset contains 18 samples belonging to two different cell lines (PACA44 and T3M4) of control (untreated) and drug-treated pancreatic ductal carcinoma cells. The aim of this work was to obtain the correct classification of the 18 samples, using the Legendre moments as discriminant variables. For each image the Legendre moments up to a maximum order of 100 were computed. The stepwise linear discriminant analysis (LDA) was performed in order to select the moments with the highest discriminating power. The results demonstrate that the Legendre moments can be successfully applied for fast classification purposes and similarity analysis.     

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.     

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.     

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.     

Prototype for integrated two-dimensional gel electrophoresis for protein separation

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h.     

Towards high performance two-dimensional gel electrophoresis using ultrazoom gels

Proteomics is the analysis of protein expression in cells or tissues, e.g., to study cellular processes at the molecular level. Ultimately, a proteome analysis should encompass most if not all protein species in a biological sample, including those present in low copy numbers. We are developing two-dimensional gel electrophoresis technology by applying narrow pH range ultrazoom gels to enhance resolution and to improve the detection of low abundance proteins. Ultrazoom gels in the acidic pH range allow the detection of proteins down to 300 copies per cell of a B-lymphoma cell line. Protein separation in the alkaline pH range, however, still requires optimization, especially in conjunction with high sample loads.     

A novel droplet-tap sample-loading method for two-dimensional gel electrophoresis

A novel procedure, droplet-tap mode, has been devised for sample application for two-dimensional gel electrophoresis (2DE) expression profiles. The sample was loaded by evenly distributed tapping of droplets of the sample on to the rehydration buffer (RB) and then lowering the strip on to the solution surface. At normal loading concentrations, the number of spots obtained was increased by approximately one-third by this new approach compared with the rehydration loading procedure. The method also resulted in significantly improved resolution compared with cup loading when high concentrations of proteins were present, indicating its potential usefulness in micropreparative separation. In addition, recovery of the proteins confirmed that protein uptake was enhanced by use of this method. By enabling improved performances in 2DE, the proposed procedure has much potential for sample loading to meet the requirements of global proteome analysis.Electronic Supplementary Material Electronic Supplementary Material found in     

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.     

A multivariate spot filtering model for two-dimensional gel electrophoresis

Image segmentation plays an important role in the automatic analysis of protein spots in 2-DE. Using image segments representing protein spots, the amount of protein in each segment can be quantified, and corresponding segments can be matched and compared for multiple gels. However, the common presence of image segments caused by noise and unwanted artefacts highly disturb the analysis and comparison of the gels. Common sources of such artefacts are cracks in the gel surface, fingerprints, dust and other pollutions. It would be advantageous to remove these unwanted artefacts during or after the segmentation procedure. To achieve this task a multivariate spot filtering model is developed using image segments from a gel segmentation. Parameters in the model are based on texture, shape and intensity measurements of the image segments. The model successfully managed to separate segments caused by noise, artefacts and cracks from image segments representing true protein spots. The classification method used is discriminant partial least squares regression.     

Ongoing development of two-dimensional polyacrylamide gel electrophoresis data standards

We present an approach toward standardizing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) data in support of developing a globally relevant proteomics consensus in order to provide more efficient database querying and data comparisons through the establishment of the necessary definitions and interdisciplinary reference fields for both the 2-D PAGE community, particularly in the proteomics area, and the clinical and experimental biological research communities, in general. This article covers the need for unifying the 2-D PAGE data through a common data repository, and its usefulness in data standards and data interoperability.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.