The MELANIE project: from a biopsy to automatic protein map interpretation by computer.

The goals of the MELANIE project are to determine if disease-associated patterns can be detected in high resolution two-dimensional polyacrylamide gel electrophoresis (HR 2D-PAGE) images and if a diagnosis can be established automatically by computer. The ELSIE/MELANIE system is a set of computer programs which automatically detect, quantify, and compare protein spots shown on HR 2D-PAGE images. Classification programs help the physician to find disease-associated patterns from a given set of two-dimensional gel electrophoresis images and to form diagnostic rules. Prototype expert systems that use these rules to establish a diagnosis from new HR 2D-PAGE images have been developed. They successfully diagnosed cirrhosis of the liver and were able to distinguish a variety of cancer types from biopsies known to be cancerous.     

Database and search techniques for two-dimensional gel protein data: a comparison of paradigms for exploratory data analysis and prospects for biological modeling

Two-dimensional (2-D) polyacrylamide gel electrophoresis can detect thousands of polypeptides, separating them by apparent molecular weight (Mr) and isoelectric point (pI). Thus it provides a more realistic and global view of cellular genetic expression than any other technique. This technique has been useful for finding sets of key proteins of biological significance. However, a typical experiment with more than a few gels often results in an unwiedly data management problem. In this paper, the GELLAB-II system is discussed with respect to how data reduction and exploratory data analysis can be aided by computer data management and statistical search techniques. By encoding the gel patterns in a "three-dimensional" (3-D) database, an exploratory data analysis can be carried out in an environment that might be called a "spread sheet for 2-D gel protein data". From such databases, complex parametric network models of protein expression during events such as differentiation might be constructed. For this, 2-D gel databases must be able to include data from other domains external to the gel itself. Because of the increasing complexity of such databases, new tools are required to help manage this complexity. Two such tools, object-oriented databases and expert-system rule-based analysis, are discussed in this context. Comparisons are made between GELLAB and other 2-D gel database analysis systems to illustrate some of the analysis paradigms common to these systems and where this technology may be heading.     

Exploratory data analysis groupware for qualitative and quantitative electrophoretic gel analysis over the Internet-WebGel

Many scientists use quantitative measurements to compare the presence and amount, of various proteins and nucleotides among series of one- and two-dimensional (1-D and 2-D) electrophoretic gels. These gels are often scanned into digital image files. Gel spots are then quantified using stand-alone analysis software. However, as more research collaborations take place over the Internet, it has become useful to share intermediate quantitative data between researchers. This allows research group members to investigate their data and share their work in progress. We developed a World Wide Web group-accessible software system, WebGel, for interactively exploring qualitative and quantitative differences between electrophoretic gels. Such Internet databases are useful for publishing quantitative data and allow other researchers to explore the data with respect to their own research. Because intermediate results of one user may be shared with their collaborators using WebGel, this form of active data-sharing constitutes a groupware method for enhancing collaborative research. Quantitative and image gel data from a stand-alone gel image processing system are copied to a database accessible on the WebGel Web server. These data are then available for analysis by the WebGel database program residing on that server. Visualization is critical for better understanding of the data. WebGel helps organize labeled gel images into montages of corresponding spots as seen in these different gels. Various views of multiple gel images, including sets of spots, normalization spots, labeled spots, segmented gels, etc. may also be displayed. These displays are active and may be used for performing database operations directly on individual protein spots by simply clicking on them. Corresponding regions between sets of gels may be visually analyzed using Flicker-comparison (Electrophoresis 1997, 18, 122-140) as one of the WebGel methods for qualitative analysis. Quantitative exploratory data analysis can be performed by comparing protein concentration values between corresponding spots for multiple samples run in separate gels. These data are then used to generate reports on statistical differences between sets of gels (e.g., between different disease states such as benign or metastatic cancers, etc.). Using combined visual and quantitative methods, WebGel can help bridge the analysis of dissimilar gels which are difficult to analyze with stand-alone systems and can serve as a collaborative Internet tool in a groupware setting.     

Wheat cultivar identification by a totally automatic soft-laser scanning densitometry and computer-aided analysis of protein electropherograms

A computer-aided analysis based on soft-laser densitometry of electrophoretic patterns is described. The system has the potential to automatically identify wheat cultivars from normalized sodium dodecyl sulfate-polyacrylamide gel electropherograms. The principle encompasses autocalibration of the relative mobilities of components of the unknown pattern using standards of Mr that bracket each pattern on the pictures, automatic identification of peaks and baseline correction, and simplified estimation of the peak intensities. The algorithm provides a normalized array that can be automatically compared with a library of standard cultivar patterns in order to determine those having the highest similarity percentage. The outputs of the program are 1) relative similarity plots which show the percentage of similarity between the unknown cultivar to that of each of the common standard cultivars put in a pattern library, 2) the names of cultivars in the library having the most similar patterns and 3) computer-generated electrophoretic graphics of these cultivars. This system is not intended to identify closely related cultivars but it is particularly recommended for cultivar identification without well-trained personnel, familiar with band location and nomenclature.     

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.     

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

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

Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systems

Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. 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), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume.     

Report of workshop on cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis

A workshop entitled Cellular Protein Databases from Two-Dimensional Gel Electrophoresis was held in Atlanta, Georgia, 28 February-1 March 1987. Its purpose was to assess the status of two-dimensional gel electrophoresis of proteins as a research methodology in biological systems, particularly in the generation of cellular protein databases. The workshop participants summarized current studies on a variety of biological systems, both prokaryotic and eukaryotic. Analysis of the progress being made led to the conclusion that electrophoretic techniques, supported by automatic scanning of gel images and computer-assisted processing, analysis and matching of gel images, are now capable of generating databases of great potential value. Factors affecting the reproducibility of protein spot patterns on gels were identified, and the extent to which gel pattern variability causes difficulties in communicating results and in integrating information from different laboratories was assessed. Measures were suggested to help overcome obstacles to the generation of comprehensive cellular protein databases from the electrophoretic resolution of total cellular proteins.     

Anwendbarkeit und Anwendung von Expertensystemen in der Analytik

Summary General prerequisites of problem definition and solving by man and machine are discussed. The advantage of expert systems over books and data base systems to have a capability of understanding problems is pointed out. The chemical knowledge needs to be restructured and cast into operational terms to prepare it for formalized reasoning. Writing program routines in artificial intelligence by a chemist means wasting time and makes bad use of his chemical expertise. The commercial empty shells TWAICE, INSIGHT 2 + and 1st CLASS have been used to build expert systems in the domains of slag analysis and Karl-Fischer titrations. Examples of method selection for routine problems, compilation of new analytical methods from computer based knowledge and of strictly user oriented trouble shooting in analytical chemistry are given.     

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

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

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.     

Automatic polarographic elucidation of electrode mechanisms by means of a knowledge-based system : Part 1. Sampled d.c. polarography

A knowledge-based system has been developed for the automatic elucidation of electrochemical mechanisms. The system is based on sampled direct current (or Tast) polarography at a dropping mercury electrode as a technique for collecting experimental information and consists of a general expert system shell for the reasoning process, the specific set of rules and experimental modules. The set of rules allows the elucidation of eight relatively simple electrode reaction mechanisms fully atomatically. The computer system has been validated with chemical systems the electrochemical behaviour of which is well established. All parts of the program are written in FORTH language for Apple II microcomputers. This expert system has an open character and new rules can be added to extend the set of mechanisms that can be determined.     

Recent advances in capillary separations for proteomics

The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.     

A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels

A new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i.e. disc electrophoresis) or the separated zones (i.e. isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50, are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.     

Combination of electrophoretic techniques for comprehensive analysis of complex protein systems

Analysis of proteins in complex mixtures such as cell lysates is presently performed by two-dimensional polyacrylamide gel electrophoresis under denaturing conditions (denaturing 2-D PAGE) followed by extraction of proteins from gel pieces and structural analysis of the proteins. This type of protein analysis is contributing to the correlation of information stored in DNA sequences with the structure of the product polypeptides. However, denaturing 2-D PAGE has its own limitations and it is necessary to develop various methods of protein analysis to reconstruct the total structure and function of proteins in complex systems. This review article summarizes the work in our laboratory to explore proteins in human plasma combining various electrophoretic techniques: nondenaturing and denaturing 2-D PAGE, capillary electrophoresis, and agarose gel isoelectric focusing.     

Peptide separations by slab gel electrophoresis in pluronic F127 polymer liquid crystals

Proteomics and peptidomics could benefit from simple methods for high-resolution separation of oligopeptides analogous to slab gel electrophoresis of proteins. Gels of Pluronic F127 copolymer surfactant were investigated as media for slab gel electrophoresis of oligopeptides using a trypsin digest of myoglobin. Concentrated solutions of Pluronic F127 are fluid at low temperatures (相似文献   

Electrophoresis in the presence of gradients: I. Viscosity gradients

In many cases, the resolution provided by capillary electrophoresis systems approaches that predicted for diffusion-limited separations. Once all device-related sources of band broadening have been eliminated or minimized, only thermal diffusion remains. In principle, peaks can be sharpened using gradients of various system characteristics such as gel concentration, buffer viscosity and electric field. However, it is not clear whether this can actually increase the resolution of the system. In this article, we focus our attention on viscosity gradients and we examine both continuous and step-like variations. Our results indicate that the performance of electrophoretic systems cannot be improved by viscosity gradients. They may provide extra stacking, and thus improve the resolution, when the injection width is non-negligible. However, for the systems considered here, the best resolution is obtained when the viscosity is uniform and the stacking is entirely performed at injection. We conclude by discussing the link between these results, the fundamental laws of thermodynamics, the nature of the detection process and the importance of having nonlinear effects in nonuniform systems.