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.     

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.     

Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Hierarchical grid transformation for image warping in the analysis of two-dimensional electrophoresis gels

Two-dimensional electrophoresis is a widely used method for separating a large number of proteins from complex protein mixtures and for revealing differential patterns of protein expressions. In the computer-assisted proteome research, the comparison of protein separation profiles involves several heuristic steps, ranging from protein spot detection to matching of unknown spots. An important prerequisite for efficient protein spot matching is the image warping step, where the geometric relationship between the gel profiles is modeled on the basis of a given set of known corresponding spots, so-called landmarks, and the locations of unknown spots are predicted using the optimized model. Traditionally, polynomial functions together with least squares optimization has been used, even though this approach is known to be incapable of modeling all the complex distortions inherent in electrophoretic data. To satisfy the need of more flexible gel distortion correction, a hierarchical grid transformation method with stochastic optimization is presented. The method provides an adaptive multiresolution model between the gels, and good correction performance in the practical cross-validation tests suggests that automatic warping of gel images could be based on this approach. We believe that the proposed model also has significance in the ultimate comparison of corresponding protein spots since the matching process should benefit from the closeness of the true spot pairs.     

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.     

A technique for electrophoresis in multiple-concentration agarose gels

A technique has been developed for embedding several agarose gels (running gels), each of a different agarose concentration, within a single 1.5% agarose slab. Equal portions of a sample were placed at the origin of each running gel and were simultaneously subjected to electrophoresis. Protein within the running gels was detected by staining with Coomassie blue; 0.2% gels were the least concentrated gels that were stained without gel breakage. Using the above technique, the dependence of electrophoretic mobility on agarose concentration has been measured for bacteriophage T7 capsids and a capsid dimer.     

The substructure of agarose gels as prepared for electrophoresis

Gelled agarose was observed by freeze-fracture and electron microscopy. Aggregated double helices of agarose form a fibrous network that showed conformational variations over a range of agarose concentrations (0.3–2%). Diameters of fibers tend to increase with rising concentrations and the appearance of protuberances or cul-de-sacs in the network increases with falling concentration.     

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.     

Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels

An extensive series of experiments has been performed to study the mobility of DNA fragments ranging in size from 2.0 to 48.5 kilobose pairs. By varying the agarose concentration in the gels and the electric field strength, three DNA electrophoresis regimes were clearly identified: the Ogston regime (small DNA fragments in large pores of agarose), the reptation regime without DNA chain stretching (small pores of agarose and weak electric fields), and the reptation regime with DNA chain stretching (small pores of agarose, strong electric fields, and large DNA fragments). Here we report on the experimental identification of these regimes and on the conditions governing the transition between each of them. The onset of reptation and of stretching of DNA chains in gel electrophoresis are described quantitatively for the first time, and a phase diagram for the dynamics of DNA during electrophoresis is presented.     

Improvements in procedures for electrophoresis in dilute agarose gels

Procedures have been developed for performing electrophoresis in agarose gels with agarose concentrations as low as 0.035%. Using these procedures, agarose gel electrophoresis of the following has been performed: (a) bacteriophage T7 missing its tail fibers; no detectable sieving of this spherical particle (radius = 30.1 nm) occurred below 0.075% agarose, (b) duplex DNAs with molecular weights between 26.5 × 10⁶ and 110 × 10⁶.     

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     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Proteomic tools for the analysis of transient interactions between metalloproteins

Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.     

Synchrotron radiation SAXS of 11S soya globulin

Small-angle X-ray scattering using the Daresbury synchrotron source has been employed to obtain scattering curves from a 5% solution of the 11S soya globulin. The high intensity of the source allowed exposure times to be reduced by up to 1000 times compared with those for a conventional X-ray generator. Submaxima at higher angles were recorded which have not been reported previously. This improved resolution appears to result from reduced aggregation and/or denaturation of the protein due to the very short exposure times. Such detail in the scattering curve should be of importance for structural modelling of the proteins, particularly in the case of the 11S soya globulin for which intact individual subunits cannot be isolated.