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.     

A two-dimensional electrophoresis-related laboratory information processing system: spot matching.

An approach for the computer-assisted analysis of two-dimensional gels has been developed as a part of our laboratory information processing system (LIPS). This approach relies in part on an algorithm for the pairwise matching of protein spots. The matching process initially matches spots based on a cross-correlational measure of how well neighboring spots align. While this first pass correctly determines most spot correspondences and noncorrespondences, it can make errors. Higher accuracy is obtained by monitoring the consistency of spot match decisions in a second pass, which demands that neighboring spot pairs that align spatially must also have been found to match in the first pass. Pairwise comparisons of gels are combined into n-way comparisons by matching spot lists of gels to "master" gel spot lists, which in turn are matched to higher level masters, resulting in a hierarchy of matched spots. After each pairwise match the results are reviewed and corrected with the assistance of a graphical match-editor. Results are given for 19 single-cell-derived lymphoid clones in which the presence of a mutation had previously been established, each processed in duplicate. Only one of 46 spot changes failed to be detected, which demonstrates that the strategy is sensitive and efficient for detecting qualitative spot differences.     

Fast automatic registration of images using the phase of a complex wavelet transform: application to proteome gels

Image registration describes the process of manipulating a distorted version of an image such that its pixels overlay the equivalent pixels in a clean, master or reference image. The need for it has assumed particular prominence in the analysis of images of electrophoretic gels used in the analysis of protein expression levels in living cells, but also has fundamental applications in most other areas of image analysis. Much of the positional information of a data feature is carried in the phase of a complex transform, so a complex transform allows explicit specification of the phase, and hence of the position of features in the image. Registration of a test gel to a reference gel is achieved by using a multiresolution movement map derived from the phase of a complex wavelet transform (the Q-shift wavelet transform) to dictate the warping directly via movement of the nodes of a Delaunay-triangulated mesh of points. This warping map is then applied to the original untransformed image such that the absolute magnitude of the spots remains unchanged. The technique is general to any type of image. Results are presented for a simple computer simulated gel, a simple real gel registration between similar "clean" gels with local warping vectors distributed about one main direction, a hard problem between a reference gel and a "dirty" test gel with multi-directional warping vectors and many artifacts, and some typical gels of present interest in post-genomic biology. The method compares favourably with others, since it is computationally rapid, effective and entirely automatic.     

A new method for assigning common spot boundaries for multiple gels in two-dimensional gel electrophoresis

The benefits of defining common spot boundaries when several gels from 2-DE are compared and analyzed have lately been stressed by both commercial software producers and users of this software. Though the importance of common spot boundaries is clearly stated, few reports exist that target this issue explicitly. In this study a method for defining common spots boundaries is developed, called the spot density method. The method consists of the following steps: segmentation and spot identification on each individual gel, transferring the spot-center coordinates for all gels onto a single new gel, collecting spot centers clustered together in the new gel and finally assigning pixels and new spot boundaries based on the spots in each cluster. The method is compared to a synthetic gel approach, and validated by visual inspection of three representative areas in the gels. The gel images need to be aligned prior to segmentation and spot identification, but the method can be used regardless of the choice of segmentation procedure. This makes the method an easy extension to existing methods for spot identification and matching. Conclusions based on the visual inspection are that the spot density method identifies partly overlapping spots and low-intensity spots better than the synthetic gel approach.     

GELANAL, a personal computer-program to compare protein patterns on two-dimensional polyacrylamide gels

Comparing and analyzing a series of two-dimensional gels by hand is troublesome and subjective. So far a number of systems for automatic analysis have been developed on mainly mainframe computers, using complex algorithms. This paper presents an inexpensive system, based on a simple Pascal program, to compare individual spots on two-dimensional gels using an IBM or compatible personal computer in a qualitative way. The accuracy of the method is demonstrated by comparing two patterns of the same extract from different runs.     

Wheat gliadin: digital imaging and database construction using a 4-band reference system of agarose isoelectric focusing patterns.

An isoelectric focusing method using thin-layer agarose gel has been developed for wheat gliadin. Using flat-bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat-bed scanner, a 4-band reference system using isoelectric points was devised. Software enables separated bands to be assigned pI values based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for pI interval required for a match, and number of best matches.     

A review of the CLIP system for the quantitative analysis of two-dimensional electrophoresis gels

This paper reviews the CLIP image processing system for the complete analysis of two-dimensional electrophoresis images. The analysis problem for two-dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two-dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation.     

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.     

Computerized methods for analyzing two-dimensional agarose gel electropherograms

Previous methods interpret zonal or polydisperse gel patterns of two-dimensional Serwer-type gels in terms of size and free mobility (surface net charge density). These two parameters have been determined for each component without quantitatively measuring the abundance of the components. The present study advances these previous methods by determining the relative concentration of each component by computer evaluation of densitometrically analyzed gel patterns. Suitable procedures and their underlying algorithms are presented. The mathematical routines are implemented in a user-friendly software package, called GelFit and designed for a Macintosh personal computer. The program input consists of digitized images of gel staining patterns exemplified by those obtained from electrophoresis of native subcellular-sized particles. The data are processed through the following steps: (i) Noise reduction and calibration. (ii) Geometrical transformation of the pattern onto a rectangular size/free mobility coordinate system using rationales of the extended Ogston model. (iii) Analysis of the transformed image to determine density maxima, density profiles along iso-free-mobility or iso-size lines, curve fitting of one-dimensional profiles or two-dimensional surfaces using Gaussian functions and curve stripping of surfaces to determine the possible number of particle populations.     

An improved pixel-based approach for analyzing images in two-dimensional gel electrophoresis

An improved pixel-based approach for analyzing 2-DE images is presented. The key feature of the method is to create a mask based on all gels in the experiment using image morphology, followed by multivariate analysis on the pixel level. The method reduces the impact of noise and background by identifying regions in the image where protein spots are present, but make no assumption on individual spot boundaries for isolated spots. This makes it possible to detect significant changes in complex regions, and visualize these changes over multiple gels in an easy way. False missing values and spot volumes caused by imposing erroneous spot boundaries are thus circumvented. The approach presented gives improved pixel-based information from the gels, and is also an alternative to existing methods for data-reduction, significance testing and visualization of 2-DE data. Results are compared with software using a common spot boundary approach on an experiment consisting of 35 full size gel images. Gel alignment is required before analysis.     

Two-dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases

A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.     

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.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

Somatic mutation studies in human lymphoid cells: the detectability of quantitative genetic variants in two-dimensional gels

The feasibility of detecting quantitative genetic variants based on a decrease in the integrated intensity of polypeptide spots in two-dimensional polyacrylamide gels of human lymphoblastoid cell clones was investigated. A battery of 65 spots on 115 gels was studied to determine the distribution of quantitative measures for spots where no mutation had occurred. The corresponding distribution for spots which have decreased integrated intensity as a result of a mutation at one of two alleles coding for the spot was investigated by quantitating spots for which mutations were known to have occurred. These two distributions allowed the estimation of the rates of false positive and false negative errors for any particular strategy aimed at detecting null mutations, and thus provides a basis for the design of efficient strategies. Our silver stained gels have sufficient reproducibility of spot integrated intensities so that, for situations in which the mutation rate is relatively high, it is practical to monitor a subset of spots for null variants using the same digitized images as are used to detect structural variants.     

Quantitative comparison and evaluation of two commercially available,two-dimensional electrophoresis image analysis software packages,Z3 and Melanie

While a variety of software packages are available for analyzing two-dimensional electrophoresis (2-DE) gel images, no comparisons between these packages have been published, making it difficult for end users to determine which package would best meet their needs. The goal here was to develop a set of tests to quantitatively evaluate and then compare two software packages, Melanie 3.0 and Z3, in three of the fundamental steps involved in 2-DE image analysis: (i) spot detection, (ii) gel matching, and (iii) spot quantitation. To test spot detection capability, automatically detected protein spots were compared to manually counted, "real" protein spots. Spot matching efficiency was determined by comparing distorted (both geometrically and nongeometrically) gel images with undistorted original images, and quantitation tests were performed on artificial gels with spots of varying Gaussian volumes. In spot detection tests, Z3 performed better than Melanie 3.0 and required minimal user intervention to detect approximately 89% of the actual protein spots and relatively few extraneous spots. Results from gel matching tests depended on the type of image distortion used. For geometric distortions, Z3 performed better than Melanie 3.0, matching 99% of the spots, even for extreme distortions. For nongeometrical distortions, both Z3 and Melanie 3.0 required user intervention and performed comparably, matching 95% of the spots. In spot quantitation tests, both Z3 and Melanie 3.0 predicted spot volumes relatively well for spot ratios less than 1:6. For higher ratios, Melanie 3.0 did much better. In summary, results suggest Z3 requires less user intervention than Melanie 3.0, thus simplifying differential comparison of 2-DE gel images. Melanie 3.0, however, offers many more optional tools for image editing, spot detection, data reporting and statistical analysis than Z3. All image files used for these tests and updated information on the software are available on the internet (http://www.umbc.edu/proteome), allowing similar testing of other 2-DE image analysis software packages.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.