The role of bioinformatics in two-dimensional gel electrophoresis

Over the last two decades, two-dimensional electrophoresis (2-DE) gel has established itself as the de facto approach to separating proteins from cell and tissue samples. Due to the sheer volume of data and its experimental geometric and expression uncertainties, quantitative analysis of these data with image processing and modelling has become an actively pursued research topic. The results of these analyses include accurate protein quantification, isoelectric point and relative molecular mass estimation, and the detection of differential expression between samples run on different gels. Systematic errors such as current leakage and regional expression inhomogeneities are corrected for, followed by each protein spot in the gel being segmented and modelled for quantification. To assess differential expression of protein spots in different samples run on a series of two-dimensional gels, a number of image registration techniques for correcting geometric distortion have been proposed. This paper provides a comprehensive review of the computation techniques used in the analysis of 2-DE gels, together with a discussion of current and future trends in large scale analysis. We examine the pitfalls of existing techniques and highlight some of the key areas that need to be developed in the coming years, especially those related to statistical approaches based on multiple gel runs and image mining techniques through the use of parallel processing based on cluster computing and the grid technology.     

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.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Quantitative protein profiling using two-dimensional gel electrophoresis,isotope-coded affinity tag labeling,and mass spectrometry

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.     

Identification of proteins overexpressed in papillary thyroid tumors

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.     

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.     

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2-3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3-5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.     

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.     

A new two-dimensional gel electrophoresis system for the analysis of complex protein mixtures: Application to the ribosome of E. coli

A new method for two-dimensional polyacrylamide gel electrophoresis of proteins is described. The method, illustrated here by its application for the analysis of ribosomal proteins of E. coli, has a high resolving power. The proteins S15 and S16 can be resolved either following alkylation or under reducing conditions. This was not possible with urea gel systems previously employed. The method should be advantageous in the identification of the components of dimers formed with the reagent methyl 4-mercaptobutyrimidate. An additional advantage of the new method is that both dimensions are run at an acidic pH. For ribosomal proteins it is therefore unnecessary to either polymerize the protein sample in the middle of the first dimension disc gel or to electrophorese two samples with opposite polarity.     

Sample preparation for two-dimensional gel electrophoresis

The choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two-dimensional gel result in terms of quality and protein species distribution. The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two-dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since protein staining is becoming increasingly incorporated into the sample preparation procedure, we describe the principles and applications of current (and future) pre-electrophoretic labelling methods.     

Comparative study of ribosomal proteins from Yersinia pestis and Escherichia coli: amino acid composition and electrophoretic mobility

The techniques of electrophoresis in polyacrylamide gel and amino acid analysis permitted to find the slight difference in the composition of ribosomal proteins from Yersinia pestis and Escherichia coli cells. Ribosomal proteins were mapped and classified on the basis of two-dimensional electrophoresis data. Protein "X" was registered in the total ribosomal protein due to its separation in course of ribosomal subunits dissociation.     

Rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis

Identification of qualitative and/or quantitative protein expression differences as well as characterization of specific cell proteomes would further advance molecular cell biology research. Today, one of the most commonly used tools for proteome analysis is two-dimensional gel electrophoresis. Although this technology is informative, it is extremely cumbersome, time-consuming and lacks automation and proper reproducibility. In this paper, we propose an automated separation/detection system capable of rapid two-dimensional analysis of proteins by ultra-thin layer gel electrophoresis with real time imaging of the separated components, using fiber optics based laser induced fluorescence technology. The approach is based on electric field mediated separation in capillary dimensions, along with noncovalent, "in migratio" fluorescent staining methodology. The advantage of the technology discussed over existing techniques is its simplicity, speed and good detection sensitivity.     

Recent advances in gel-based proteome profiling techniques

This review focuses on recent developments in gel-based proteomics techniques. By combining traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoretic techniques with recent advances in protein labeling using different classes of molecules (i.e., fluorescent dyes, chemical probes, radioisotopes), new technologies have been developed that allow for high-throughput studies of proteins at the whole-proteome scale.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.     

Proteomic analyses of amniotic fluid: potential applications in health and diseases

Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.     

Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via naOH for two-dimensional electrophoresis

In preparing intracellular microbial samples for one- or two-dimensional electrophoresis, trichloroacetic acid (TCA) precipitation is frequently used to remove interfering compounds. Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is presented. Precipitated proteins are pretreated with 0.2 M NaOH for less than 5 min, followed by addition of standard sample solubilization buffer (SSSB). When compared to solubilization with SSSB alone, NaOH pretreatment of TCA-precipitated intracellular protein from Aspergillus oryzae and Escherichia coli shows an approximate 5-fold increase in soluble protein. In addition, two-dimensional gel electrophoresis on resolubilized proteins shows an equivalent number of proteins in samples with and without NaOH pretreatment.     

Non-covalent and covalent protein labeling in two-dimensional gel electrophoresis

Over the past decades, several sensitive post-electrophoretic stains have been developed for an identification of proteins in general, or for a specific detection of post-translational modifications such as phosphorylation, glycosylation or oxidation. Yet, for a visualization and quantification of protein differences, the differential two-dimensional gel electrophoresis, termed DIGE, has become the method of choice for a detection of differences in two sets of proteomes. The goal of this review is to evaluate the use of the most common non-covalent and covalent staining techniques in 2D electrophoresis gels, in order to obtain maximal information per electrophoresis gel and for an identification of potential biomarkers. We will also discuss the use of detergents during covalent labeling, the identification of oxidative modifications and review influence of detergents on finger prints analysis and MS/MS identification in relation to 2D electrophoresis.     

Proteins associated with disease and clinical course in pancreas cancer: a proteomic analysis of plasma in surgical patients

New biomarkers for pancreas cancer are needed to improve its detection and management. We surveyed the plasma of patients undergoing surgical resection to identify proteins which change in abundance after complete resection of tumor. Using longitudinally collected specimens from surgical patients, we control for normal inter-individual variation which can confound cross-sectional analysis. Recent refinements in two-dimensional gel electrophoresis allowed us to quantify changes in low abundance plasma proteins with precision. To circumvent the traditional limitations of image analysis in comparing two-dimensional gels, we used fluorometric two-dimensional difference gel electrophoresis to resolve the proteins from pre- and post-surgical plasma from each patient on one physical gel. Furthermore, we increased the ability of our assay to detect low-abundance proteins by depleting the plasma of 12 high-abundance proteins with a multi-affinity column. Informative protein spots from 20 plasma samples across 10 patients were submitted for identification with mass-spectrometry. We identified a group of proteins which change consistently in plasma following complete resection of pancreas tumor. Furthermore, we identified proteins which correlate with post-surgical rapid recurrence of disease. With further identification and validation, the candidate biomarkers which we identify in this study may prove to be useful in the diagnosis, management and prognostication of patients with pancreas cancer.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.