Quantitative and qualitative measure of intralaboratory two-dimensional protein gel reproducibility and the effects of sample preparation, sample load, and image analysis

We investigate one approach to assess the quantitative variability in two-dimensional gel electrophoresis (2-DE) separations based on gel-to-gel variability, sample preparation variability, sample load differences, and the effect of automation on image analysis. We observe that 95% of spots present in three out of four replicate gels exhibit less than a 0.52 coefficient of variation (CV) in fluorescent stain intensity (% volume) for a single sample run on multiple gels. When four parallel sample preparations are performed, this value increases to 0.57. We do not observe any significant change in quantitative value for an increase or decrease in sample load of 30% when using appropriate image analysis variables. Increasing use of automation, while necessary in modern 2-DE experiments, does change the observed level of quantitative and qualitative variability among replicate gels. The number of spots that change qualitatively for a single sample run in parallel varies from a CV = 0.03 for fully manual analysis to CV = 0.20 for a fully automated analysis. We present a systematic method by which a single laboratory can measure gel-to-gel variability using only three gel runs.     

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

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

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 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.     

Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis)

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.     

Cardiac muscle protein analysis by high-resolution and microscale two-dimensional gel electrophoresis

An improved method of two-dimensional gel electrophoresis of small muscle samples is described. Isoelectric focusing of cardiac whole muscle homogenate in agarose gels containing urea and detergent has a markedly increased resolution. Equilibration of the first-dimensional gels with detergent before application to the second-dimensional gels is unnecessary in this system. By applying this method to rat cardiac whole muscle, high-molecular weight proteins, such as myosin heavy chains, are focused on the first-dimensional gels and, in addition, minor components are resolved on the second-dimensional gels, without loss during equilibration with detergent. The two-dimensional electrophoretic patterns of rat cardiac whole muscle obtained with this method reveal numerous clearly separated spots. By analyzing the two-dimensional electrophoretic patterns of rat cardiac whole muscle and various rat cardiac fractions, and by staining the calcium-binding proteins with "Stains-all", we identified some cardiac muscle components, such as myosin heavy and light chains, actin, tropomyosin, and troponin C, but additional work is required to identify the remaining spots. The two-dimensional electrophoretic system described here makes possible the effective resolution of whole cardiac muscle homogenate from small samples, and looks promising as an aid to muscle research.     

中国小型猪心肌梗死模型的比较蛋白质组学研究

利用二维电泳(2DE)分离中国小型猪心肌梗死模型的正常与梗死心肌组织的蛋白提取液, 采用 PDQuest 软件对比分析了两种心肌组织在pH=5─8范围内的2DE谱图. 正常心肌组织检出851个蛋白点, 梗死组织检出1 032个蛋白点. 发现13个蛋白质点只在小型猪的正常心肌组织中表达, 而有14个蛋白质点只在梗死心肌组织中表达. 另外, 还有49个蛋白点在两种组织中表达量上有显著性变化(P<0.05), 选择进行质谱分析其中11个蛋白点, 成功地鉴定出7种蛋白, 蛋白功能分析结果表明, 这些蛋白的差异表达与心肌梗死过程相关.     

Influence of image‐analysis software on quantitation of two‐dimensional gel electrophoresis data

Image analysis of two‐dimensional gels is a crucial step in a proteomic workflow and has a direct impact on obtained qualitative and quantitative data. Since the analysis is a complex process and creates large data amounts, the use of a respective software is inevitable. There are only a few papers published addressing the issue of analysis‐based variance; therefore, our aim was to highlight the discrepancy of received results when different commercially available image‐tools are used for gel analysis especially in terms of comparability of the obtained outcome when the same digital image set is used. A set of six gels (three replicates per group) of real‐life samples was created and examined with two different versions of PD‐Quest (Bio‐Rad) (version 6.1 and its update version 8.0) and with an external image‐tool Delta 2D (Decodon) (version 3.6). Replicate groups were analyzed and compared with each other with regard to volume ratios of a group of significantly changed spots. The study points out significant variations among results depending on the software package used, underlining the importance of a careful investigation of post‐experimental processes to receive comparable and reliable results.     

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

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

Analysis of one-dimensional gels and two-dimensional Serwer-type gels on the basis of the extended Ogston model using personal computers

This report presents the stand-alone computer application ELPHOFIT, a software package for the analysis of gel electrophoretic data based on Ferguson plots. Either conventional one-dimensional gels or two-dimensional agarose gels (Serwer-type) can be evaluated. Special emphasis is on the latter gel type, which has been applied previously for the separation of DNA, intact viruses and polydisperse meningitis vaccines. ELPHOFIT is designed for Macintosh PCs and for the IBM XT, AT, PS/2 and compatibles. The program operates interactively with the user, who determines the course of evaluation. Data input is in the format of files providing values of gel electrophoretic migration distances or particle mobility (absolute or relative). Data processing involves a simultaneous least-square curve fitting algorithm (Newton-Gauss, Marquardt-Levenberg) which uses equations derived from the extended Ogston model. Functions are fit to the database by adjusting their variables, representing physical parameters of the gel and the electrophoresed particle. The program output consists of tables and graphics accompanied by an explanatory text providing the following information: (i) radius and free mobility of the electrophoresed particle, (ii) fiber radius, length and volume, mean or median pore radius of the gel, (iii) linear Ferguson plots, (iv) iso-free-mobility/iso-size nomogram for two-dimensional gels, (v) confidence ellipses, (vi) required parameters for image processing program GELFIT and (vii) goodness-of-fit and other statistical parameters, such as standard errors, dependency values, root-mean-square (RMS) error and determination coefficient. Other features of the program are (i) simulation of Serwer-type two-dimensional electrophoresis, (ii) standardization according to size, or size and free mobility, (iii) the conversion of particle radii to molecular (or particle) weight and vice versa, (iv) interconversion of DNA size specifications, i.e. the number of base pairs and the geometric mean radii, (v) computation of gel concentration for optimal resolution of two components, (vi) option to obtain a session record, (viii) option to establish a data output file containing the information of generated graphics (IBM only) and (ix) a text editing facility, e.g., for creating data files. Graphics (Macintosh version, PICT format) and text output files (both IBM and Macintosh versions, standard ASCII format) generated by ELPHOFIT are compatible with commercially available software.     

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.     

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.     

A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell

We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis. These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions. The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection. The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots. The Protean IEF cell had the next highest number of spots detected per gel. In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel. Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed. A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed.     

Cluster analysis method in electrophoretic patterns classification

The conventional electrophoresis methods are well known techniques for protein detection and analysis of cerebrospinal fluid (CSF). Disc electrophoresis (DEP) was carried out for detection of oligoclonal IgG bands in cerebrospinal fluid (CSF) on polyacrilamide gel. However, the advance of automation has made rapid collection of large amounts of data feasible and the development of microcomputers has made sophisticated processing even of old electrophoregrams possible. Automated analysis, data storage and sophisticate data acquisition were carried out with Gel Pro Analyzer 3.1, which is specifically structured to analyze gels and elctrophoregrams: complex band pattern matching (gel variation, dendogram analysis etc.); lane relation studies (sophisticated lane database); general gel analysis (accurate molecular size, quantitative determination of protein mixture etc.). Clustering techniques have been applied for detection of intrathecal immune response. Different hierarchic cluster analysis methods such as single linkage, complete linkage, unweighted pair-group average (UPMGA) were used. In addition, other cluster characteristics such, distance matrix and Euclidean distance matrix were calculated. Pairing of electrophoresis methods and cluster image analysis, could lead to additional diagnostic information of inflammatory conditions of the central nervous system (CNS) or dysfunction of blood-CSF barrier.     

Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle

We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes.     

A multivariate spot filtering model for two-dimensional gel electrophoresis

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

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.     

Superimposing two-dimensional gels to study genetic variation in malaria parasites.

Two-dimensional polyacrylamide gel electrophoresis is a valuable tool for studying genetic variation in the human malaria parasite, Plasmodium falciparum. It involves examining the position of protein spots in gel produced from different isolates. Some spots have been seen to vary, while others have had a constant position in all isolates so far examined. These invariant spots provide a reference frame to compare variations in other spots. This paper discusses the usefulness of digital image handling, warping and superimposition in a personal computer environment. Rather than produce a fully automatic interpretation system, we show how the computer may be used as a tool for manipulating gel images, although interpretation of the gels' features remains with the human expert. Autoradiographs are scanned on a desktop scanner, and the images in digital form can be displayed on a monitor attached to a personal computer. The coordinates of the invariant spots on each of several gels are identified by the user. Each of the gels is then warped so that the invariant spots of all the gels coincide as closely as possible. The variable spots are then examined. We have used both affine warping transformations, which match the invariant spots as closely as possible, and thin plate spline transformations, which match them exactly. Colour superimposition proved a useful way of examining the gels.     

Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]