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.     

JVirGel: Calculation of virtual two-dimensional protein gels

We developed JVirGel, a collection of tools for the simulation and analysis of proteomics data. The software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. The utilization of all proteins of an organism of interest deduced from genes of the corresponding genome project in combination with the elimination of obvious membrane proteins permits the creation of an optimized calculated proteome map. The electrophoretic separation behaviour of single proteins is accessible interactively in a Java(TM) applet (small application in a web browser) by selecting a pI/MW range and an electrophoretic timescale of interest. The calculated pattern of protein spots helps to identify unknown proteins and to localize known proteins during experimental proteomics approaches. Differences between the experimentally observed and the calculated migration behaviour of certain proteins provide first indications for potential protein modification events. When possible, the protein spots are directly linked via a mouse click to the public databases SWISS-PROT and PRODORIC. Additionally, we provide tools for the serial calculation and visualization of specific protein properties like pH dependent charge curves and hydrophobicity profiles. These values are helpful for the rational establishment of protein purification procedures. The proteomics tools are available on the World Wide Web at http://prodoric.tu-bs.de/proteomics.php.     

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.     

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.     

Polyamines as cancer markers: applicable separation methods

Spermine, spermidine, putrescine and cadaverine are aliphatic amines widely spread in the human body. Their concentrations together with their acetyl conjugates increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient’s condition on multiple therapy. Various chromatographic techniques are frequently used in monitoring concentrations of di- and polyamines in cancer. Among these techniques, thin-layer chromatography and liquid chromatography using pre- or postcolumn derivatization, separating on a reversed-phase or an ion-exchange column are the most commonly used. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations. The review examines the prospects and the limitations of polyamines as cancer markers using chromatographic and electrophoretic techniques.     

Carotenoids: separation methods applicable to biological samples

Epidemiologic and clinical studies have shown that a high intake of vegetables and fruit, with consequently high intakes and circulating concentrations of carotenoids, is associated with reduced risk of cardiovascular and other chronic diseases. The antioxidant properties of carotenoids are thought to contribute to these effects. The analysis of carotenoids in plasma, foods and tissues has thus become of interest in studies examining the role of diet in chronic disease prevention and management. High-performance liquid chromatography with ultra-violet or photodiode array detection is most often employed in routine use. We review these and other current methods for carotenoid analysis and information on sample stability relevant to epidemiological studies. The carotenoids remain an important and intriguing subject of study, with relevance to prevention of several important "lifestyle-related" diseases. Research into their physiological functions and their use as dietary markers requires sensitive, accurate and precise measurement. Further advances in these methodological areas will contribute to basic, clinical and public health research into the significance of carotenoid compounds in disease prevention.     

Geometrical planning for the correction of orbital hypertelorism

Orbital hypertelorism may be associated with a variety of deformities affecting several elements of the craniofacial skeleton. Shortness of the central portion of the face represented by a wide, short nose and anterior open bite is frequently combined with the exaggerated interorbital distance. With the mobilization of the two halves of the face it is possible to approximate the orbits, simultaneously elongating the center of the face and normalizing the maxillary alveolar ridge. A technique is described to plan the operation geometrically in order to predict accurately the skeletal correction, the change of the inclination of the eye slant, and the modification of the axis of the teeth.     

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.     

Evaluation of individual protein errors in silver-stained two-dimensional gels

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error.     

Antitumor drugs possessing topoisomerase I inhibition: applicable separation methods

Separation methods for antitumor drugs capable of topoisomerase I inhibition were reviewed in this study. Camptothecin (CPT) its related analogues seemed to be promising anticancer drugs that exhibit topoisomerase I inhibition. This group of compounds contain a closed α-hydroxy-δ-lactone ring (lactone form) that can undergo reversible hydrolysis to form the open-ring form (carboxylate form). In vitro pharmacological study showed that the antitumor activity of the lactone form was higher than that of the carboxylate form. Thus a quantitative method to separate these two forms is important to evaluate the pharmacokinetics and pharmacodynamics of these compounds. Nevertheless, current separation methods are complicated by the pH-dependent instability of the lactone moiety. High-performance liquid chromatography (HPLC) coupled with fluorometric detection has been widely used for the quantitation of the drug as the intact lactone form or as the total lactone carboxylate forms in biological matrices. In this report we reviewed current applicable chromatographic techniques for further bioanalytical studies of CPT derivatives including sample preparations, HPLC columns, mobile phases and additives.     

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.     

Random genome deletion methods applicable to prokaryotes

Through their enabling of simultaneous identification of multiple non-essential genes in a genome, large-segment genome deletion methods are an increasingly popular approach to minimize and tailor microbial genomes for specific functions. At present, difficulties in identifying target regions for deletion are a result of inadequate knowledge to define gene essentiality. Furthermore, with the majority of predicted open reading frames of completely sequenced genomes still annotated as putative genes, essential or important genes are found scattered throughout the genomes, limiting the size of non-essential segments that can be safely deleted in a single sweep. Recently described large-segment random genome deletion methods that utilize transposons enable the generation of random deletion strains, analysis of which makes identification of non-essential genes less tedious. Such and other efforts to determine the minimum genome content necessary for cell survival continue to accumulate important information that should help improve our understanding of genome function and evolution. This review presents an assessment of technological advancements of random genome deletion methods in prokaryotes to date.     

Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels

In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete chromosomal locations. At each initiation site, a single small replication bubble is generated, which subsequently expands at Y-like replication forks. We wanted to know whether other eukaryotic organisms utilize similar initiation mechanisms. For this purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.     

Diagnostic value of urinary orotic acid levels: applicable separation methods

Urinary orotic acid determination is a useful tool for screening hereditary orotic aciduria and for differentiating the hyperammonemia disorders which cannot be readily diagnosed by amino acid chromatography, thus reducing the need for enzyme determination in tissue biopsies. This review provides an overview of metabolic aberrations that may be related to increased orotic acid levels in urine, and summarises published methods for separation, identification and quantitative determination of orotic acid in urine samples. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the analysis of urinary specimens are described. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.     

Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.     

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.     

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).     

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.     

Separation methods applicable to urinary creatine and creatinine

Urinary creatinine has been analyzed for many years as an indicator of glomerular filtration rate. More recently, interest in studying the uptake of creatine as a result of creatine supplementation, a practice increasingly common among bodybuilders and athletes, has lead to a need to measure urinary creatine concentrations. Creatine levels are of the same order of magnitude as creatinine levels when subjects have recently ingested creatine, while somewhat elevated urinary creatine concentrations in non-supplementing subjects can be an indication of a degenerative disease of the muscle. Urinary creatine and creatinine can be analyzed by HPLC using a variety of columns. Detection methods include absorption, fluorescence after post-column derivatization, and mass spectrometry, and some methods have been automated. Capillary zone electrophoresis and micellar electrokinetic capillary chromatography have also been used to analyze urinary creatine and creatinine. Creatine and creatinine have also been analyzed in serum and tissue using HPLC and CE, and many of these separations could also be applicable to urinary analysis.