Microcomputer-based image analysis systems for two-dimensional electrophoresis gels

The intent of this overview is to provide the readers, especially those who are currently conducting two-dimentional electrophoresis, a basic understanding in the construction and use of microcomputer-based systems for the analysis of protein profiles generated by two-dimensional gel electrophoresis. In addition, a microcomputer-based system, employing fixed-point operations and effective algorithms, has been evaluated. The validity of this system has been demonstrated by using the two-dimensional silver-stained gels and fluorograms derived from the rat prostate. It is concluded that the present system can be used to aid the analysis of two-dimensional electrophoresis gels. An overall consideration of hardware and software components of a computer-based system is briefly discussed.     

Computer analysis of double-labeled two-dimensional electrophoresis gels

A computerized process for the automatic analysis of double-label autoradiography after two-dimensional polyacrylamide gel electrophoresis has been developed. Matching fluorographs and autoradiographs produced from gels containing 3H- and 14C-labeled proteins are digitized by a rotating drum densitometer and analyzed by the Man-computer Interactive Data Analysis System III. This system locates corresponding protein spots in the films with edge-detection algorithms, converts spot density readings to isotopic disintegrations by reference to standard curves, and computes a 3H:14C ratio for each spot in the gels. On the average, calculated ratios are accurate to approximately 9% for test strips of polyacrylamide gel containing uniform mixtures of 3H and 14C. Values obtained for two-dimensional gels containing n protein spots with a known 3H:14C ratio of 8.6 +/- 0.1 are as follows: 8.1 +/- 1.4 (n = 268), 8.8 +/- 2.1 (n = 278), 9.1 +/- 1.7 (n = 245), and 8.8 +/- 2.2 (n = 223). The computer process greatly reduces the time required to precisely compare two complex protein mixtures and has sufficient precision to detect a doubling in the biosynthesis of any individual protein.     

Profile matching and profile subtraction: application of computer-based image analysis system for two-dimensional electrophoresis gels

The microcomputer-based image analysis system IB-1000 (developed by Indiana Biotech, Highland, IN) for two-dimensional electrophoresis gels has been described previously (9). It allows the user to compare protein spots between two profiles and identify those spots that are commonly shared in both profiles. This report describes two applications of the system's global comparison routine-profile matching and profile subtraction. This application is able to subtract commonly shared spots from one profile, creating a new profile made up by the unmatched spots in the other profile. These applications can be employed in a large variety of research projects.     

Alignment of two-dimensional electrophoresis gels

Two-dimensional electrophoresis is a major separating technique for proteins in proteomics. Alignment of gel images is critical for intra-laboratory or even more difficult inter-laboratory gel comparisons. In the paper, we propose a novel iterative closest point (ICP) method for 2D-gel electrophoresis image alignment. The paper seeks to introduce an information theoretic measure as one part of distance metric to gel image alignment. We combine intensity information of spots with geometric information of landmarks by applying information potential idea. The proposed method has been applied to both synthetic and real gel images accessible in public 2D-electrophoresis gel protein databases. The high accuracy and robustness of the algorithm indicate that it is promising for gel image alignment.     

GENOCOP algorithm and hierarchical grid transformation for image warping of two dimensional gel eletrophoretic maps

Hierarchical grid transformation is a powerful approach to SDS 2DPAGE maps warping. The hierarchy of the warping transformation is able to model both global and local deformations of the gels and the algorithm can be stopped when a certain degree of accuracy in the image alignment is obtained. The numerical optimization of the position of the nodes of the grid that are responsible for the image warping is a multivariate task that can be solved efficiently using Genetic Algorithms. The use of Genetic Algorithms ensures that an optimal position of the nodes can be defined with a low computational cost with respect to other methods. The optimal positions of the nodes of the grid can be successfully used for defining a good warping of the gels.     

Hierarchical analysis of large-scale two-dimensional gel electrophoresis experiments

Large-scale two-dimensional gel experiments have the potential to identify proteins that play an important role in elucidating cell mechanisms and in various stages of drug discovery. Such experiments, typically including hundreds or even thousands of related gels, are notoriously difficult to perform, and analysis of the gel images has until recently been virtually impossible. In this paper we describe a scalable computational model that permits the organization and analysis of a large gel collection. The model is implemented in Compugen's Z4000 system. Gels are organized in a hierarchical, multidimensional data structure that allow the user to view a large-scale experiment as a tree of numerous simpler experiments, and carry out the analysis one step at a time. Analyzed sets of gels form processing units that can be combined into higher level units in an iterative framework. The different conditions at the core of the experiment design, termed the dimensions of the experiment, are transformed from a multidimensional structure to a single hierarchy. The higher level comparison is performed with the aid of a synthetic "adaptor" gel image, called a Raw Master Gel (RMG). The RMG allows the inclusion of data from an entire set of gels to be presented as a gel image, thereby enabling the iterative process. Our model includes a flexible experimental design approach that allows the researcher to choose the condition to be analyzed a posteriori. It also enables data reuse, the performing of several different analysis designs on the same experimental data. The stability and reproducibility of a protein can be analyzed by tracking it up or down the hierarchical dimensions of the experiment.     

Minimizing variability in two-dimensional electrophoresis gel image analysis

For reliable protein identification and quantitation, it is important to minimize the variability associated with two-dimensional electrophoresis (2-DE) analysis. Since experimental factors contribute largely to the variability observed in 2-DE, most studies have focused on reducing this variability with modest concern to the variability associated with post-experimental analyses. Although often ignored, software analyses of 2-DE gel images present a considerable source of variability in the analysis of proteins. In particular, cropping of gel images prior to quantitative 2-DE analysis has been shown to contribute a significant amount of variability in image analysis. To address this problem, we propose a simple, reliable, and objective method of cropping 2-DE gel images to consequently minimize the variability in 2-DE analysis.     

Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Ultraviolet imaging densitometry of unstained gels for two-dimensional electrophoresis

A system suitable for ultraviolet imaging densitometry of two-dimensional electrophoretic gels that are unstained is described, together with its applications. A flying-spot densitometer linked with a personal computer was used for data acquisition, generation of mapping data, and image processing. Randomly distributed zones of proteins on two-dimensional gels were detected at 280 nm without being stained by two-dimensional scanning, and the densitometric value of each pixel (0.2 x 0.2 mm) was memorized by the computer, which generated a mapping pattern with density contours. The amount and densitometric value of cytochrome c had a linear relationship in the range of 2-200 micrograms. Zone locations of bovine liver proteins separated on two-dimensional gels were indicated on a map expressed in X-Y coordinates, and the pIs and molecular weights could be calculated from the map by use of pI and molecular weight markers on the same gel.     

Comparison of PDQuest and Progenesis software packages in the analysis of two-dimensional electrophoresis gels

Efficient analysis of protein expression by using two-dimensional electrophoresis (2-DE) data relies on the use of automated image processing techniques. The overall success of this research depends critically on the accuracy and the reliability of the analysis software. In addition, the software has a profound effect on the interpretation of the results obtained, and the amount of user intervention demanded during the analysis. The choice of analysis software that best meets specific needs is therefore of interest to the research laboratory. In this paper we compare two advanced analysis software packages, PDQuest and Progenesis. Their evaluation is based on quantitative tests at three different levels of standard 2-DE analysis: spot detection, gel matching and spot quantitation. As test materials we use three gel sets previously used in a similar comparison of Z3 and Melanie, and three sets of gels from our own research. It was observed that the quality of the test gels critically influences the spot detection and gel matching results. Both packages were sensitive to the parameter or filter settings with respect to the tendency of finding true positive and false positive spots. Quantitation results were very accurate for both analysis software packages.     

Multiresolution image registration for two-dimensional gel electrophoresis

In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the comparison of 2-D protein profiles, however, lies in the use of a fast and robust image matching process which is essential to the subsequent quantification procedure. To satisfy the growing demand for a robust and fully automatic method of matching 2-D gel protein separation profiles, we describe in this paper a novel registration technique based on image intensity distribution rather than selected features. The method uses a multiresolution representation of the gel profiles and exploits the fact that coarse approximations to the optimal matching can be extracted efficiently from low-resolution images. This permits the removal of misalignments at different scales in a systematic manner and the strength of the new method has been confirmed by a double blind trial of 111 2-D gel pairs. The proposed method requires neither landmarks nor an a priori image alignment, and takes about five seconds for processing a typical gel pair on a standard personal computer.     

Very-high-resolution two-dimensional gel electrophoresis of proteins using giant gels

An improved high-resolution two-dimensional gel system for separating complex protein mixtures is described that allows a threefold increase in the number of proteins detected. Like the original O'Farrell system, proteins are separated in the first dimension by isoelectric point and in the second dimension by size. The improved resolution results primarily from a 2.5-fold increase in the size of both dimensions. Although best resolution is obtained by application of <100 μg of protein containing >5 × 10⁶ cpm, as much as 150 μg of protein may be applied without appreciable loss of resolution. Useful separations may be made with up to 1.5 mg. By doubling the thickness of both dimensions, as much as 3 mg of protein can be separated into 300–400 separate peaks.     

Computer analysis of two-dimensional gels

New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels.     

A robust high-sensitivity algorithm for automated detection of proteins in two-dimensional electrophoresis gels

The automated interpretation of two-dimensional gel electrophoresisimages used in protein separation and analysis presents a formidableproblem in the detection and characterization of ill-definedspatial objects. We describe in this paper a hierarchical algorithmthat provides a robust, high-sensitivity solution to this problem,which can be easily adapted to a variety of experimental situations.The software implementation of this algorithm functions as partof a complete package designed for general protein gel analysisapplications.     

Establishing two-dimensional gels for the analysis of Leishmania proteomes

Several different sample preparation methods for two-dimensional electrophoresis (2-DE) analysis of Leishmania parasites were compared. From this work, we were able to identify a solubilization method using Nonidet P-40 as detergent, which was simple to follow, and which produced 2-DE gels of high resolution and reproducibility.     

High resolution scanning of absorbing and fluorescent electrophoresis gels using video image analysis

A low cost, microcomputer-controlled image analysis system isdescribed which scans electrophoresis gels or photographic negativeswith high resolution. Absorbing gels (e.g. with stained proteins)are analyzed using broadband or monochromatic visible light.The gel is scanned by a video camera with a macro objective;the gray level of each pixel is digitized sequentially and thevalues are stored in the computer memory. Repetitive scanningis used to average the absorption values before plotting ona digital plotter. Photographic negatives of gels and autoradiogramsare scanned using the same technique. Fluorescent, non-absorbinggels (e.g. nucleic acids stained with ethidium bromide) areanalyzed on a transilluminator with u. v. excitation radiation.The fluorescence intensity of each pixel is digitized and processedas described above. Received on June 4, 1987; accepted on July 21, 1987     

Computer analysis of two-dimensional gels.

This work identifies statistical algorithms which need to be included in analysis of two-dimensional gels for accurate determination of differential changes. Two-dimensional electrophoresis is a powerful tool for determining differential protein expression in complex mixtures, but the methodology, to date, is not producing expected results due to the degree of gel variability. The new DIGE procedure, comparing two samples in the same gel, does eliminate some of the variability introduced with gel-to-gel comparison, but still has variability due to differences in dye binding, charge, and fluorescence. Introducing quality-assurance statistical algorithms is necessary to extract meaningful data from the gels. A quality-control analysis of replicate gels needs to be performed prior to using the set in the final analysis. Increasing replicates to five from the usual three can only add greater variability. A statistical "replicate quality" gel test needs to be done on the computer gel scans, and replicates with greater than 20-30% variability should not be used. In addition, since spot intensity data are not normally distributed, spot differential analysis cannot be a t-test. The Studentized Range has been suggested as a more accurate method for calculating significant difference.     

Use of "submarine" gel electrophoresis for running multiple two-dimensional protein gels

An apparatus commonly used for the electrophoresis of submerged agarose gels was used to separate proteins in the second dimension, after isoelectric focusing in the first dimension. Multiple second-dimension gels were stacked one above the other and run horizontally, submerged in the sodium dodecyl sulfate-containing Laemmli buffer system. The reproducibility of the gels run under these conditions is remarkable and eliminates the need for individual vertical electrophoresis units for routine analysis. The units for submerged horizontal gel electrophoresis are easily made or are inexpensively available commercially.     

Using statistical image models for objective evaluation of spot detection in two-dimensional gels

Protein spot detection is central to the analysis of two-dimensional electrophoresis gel images. There are many commercially available packages, each implementing a protein spot detection algorithm. Despite this, there have been relatively few studies comparing the performance characteristics of the different packages. This is in part due to the fact that different packages employ different sets of user-adjustable parameters. It is also partly due to the fact that the images are complex. To carry out an evaluation, "ground truth" data specifying spot position, shape and intensities needs to be defined subjectively on selected test images. We address this problem by proposing a method of evaluation using synthetic images with unambiguous interpretation. The characteristics of the spots in the synthetic images are determined from statistical models of the shape, intensity, size, spread and location of real spot data. The distribution of parameters is described using a Gaussian mixture model obtained from training images. The synthetic images allow us to investigate the effects of individual image properties, such as signal-to-noise ratios and degree of spot overlap, by measuring quantifiable outcomes, e.g. accuracy of spot position, false positive and false negative detection. We illustrate the approach by carrying out quantitative evaluations of spot detection on a number of widely used analysis packages.     

The QUEST system for quantitative analysis of two-dimensional gels

The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated. Radiolabeled proteins, resolved on two-dimensional gels and detected using calibrated exposures to film, are quantified in units of disintegrations per minute or as a fraction of the total protein radioactivity applied to the gel. Spot quantitation and resolution of overlapping spots is performed by two-dimensional gaussian fitting. Pattern matching is carried out for groups of gels called matchsets, and within each matchset every gel is matched to every other gel. During the matching process, spots are automatically added to each pattern at positions where unmatched spots were detected in other patterns. This results in enhanced accuracy for both spot detection and for matching. The spot fitting procedure is repeated after matching. Tests show that up to 97% of spots in each pattern can be matched and that fewer than 1% of the spots are matched inconsistently. Approximately 2000 proteins are detected from typical gels. Of these 1600 are high quality spots. Tests to measure the coefficient of variation of spot quantitation versus spot quality show that the average coefficient of variation for high quality spots is 21%. The intensities of the detected proteins range from 4 to 20,000 ppm of total protein synthesis. The QUEST analysis system has been used to build a quantitative database for the proteins of normal and transformed REF52 cells, as presented in the accompanying reports (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5283-5298, 5299-5312).