Image analysis – Applications – Biomedical applications
Reexamination Certificate
1999-06-21
2003-10-28
Patel, Jayanti K. (Department: 2625)
Image analysis
Applications
Biomedical applications
C435S006120
Reexamination Certificate
active
06640000
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to method and apparatus for analyzing images, and more particularly to comparing related or similar images (black and white or color). The invention relates in preferred embodiments to a computer-based process, a computer-based system, a computer program, and/or a computer program product for analyzing gel electrophoresis images to identify new proteins or proteomes, and/or to compare gel images to identify quantitative or qualitative changes in proteins.
2. Related Art
Because of acidic and/or basic side chains, proteins include positively and/or negatively charged groups. The behavior of a protein in an electric field is determined by the relative numbers of these positive and/or negative charges, which in turn are affected by the acidity of the solution. At the isoelectric point (pI), the positive and negative charges are exactly balanced and the protein shows no net migration. Proteins with a pI less than about 7.5 are usually analyzed in isolelectric focusing (IFE) gels. The pH gradient in these gels is usually preformed and the sample applied at the neutral end of the gel (all acidic proteins—proteins with a pI smaller than 7.5—will be charged negatively). At the start of electrophoresis, all the negatively charged proteins will start to migrate towards the anode, i.e. through the gel. As each protein reaches its pI, the electrical forces on the protein from the cathodic and anodic sides become equal and the protein thus “focuses.” Proteins with a pI greater than 7 are best resolved on non-equilibrium pH gradient electrophoresis (NEPHGE) gels. Hence the proteins are applied again at the neutral pH region and all basic proteins (with a pI greater than 7) will be charged positively. During electrophoresis, they will therefore move towards the cathode, i.e., through the gel.
Obviously, the same separation could be obtained in an immobilized pH gradient electrophoresis gel system (IPG) where the pH gradient is covalently bound to the support matrix (polyacrylamide). Different proteins have different proportions of acidic and/or basic side chains, and hence have different isoelectric points. In a solution of a particular hydrogen ion concentration (pH), some proteins move toward a cathode and others toward an anode. Depending upon the size of the charge as well as upon molecular size and/or shape, different proteins move at different speeds. This difference in behavior in an electric field is the basis of the electrophoresis method of separation and/or analysis of proteins.
Two-dimensional gel electrophoresis (2DGE) is a particularly effective tool for analyzing proteins. Cell extracts from any prokaryotic or eukaryotic cell are put onto a gel, and the individual proteins are separated first by the pI (first dimension) and then by size (second dimension). The result is a characteristic picture of as many as 1000 to 5000 spots, each usually a single protein. Resolution is improved by increasing gel composition or size, and/or by enhancing the sensitivity through the use of radiochemical methods, silver staining, and/or the reduction in thickness of the gels to 1.5 mm or less. Jungblut et al. have reported up to 5000 protein spots of mouse brain on gels of size 23×30 cm (
Journal of Biotechnology,
41 (1995) 111-120).
High resolution 2DGE has been used for analyzing basic as well as acidic proteins. Isoelectric focusing (IEF) in the first dimension can be combined with sodium dodecyl sulfate (SDS) gel electrophoresis in the second dimension (IEF-SDS). Alternatively, nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension can be combined with SDS gel electrophoresis in the second dimension (NEPHGE-SDS). Such procedures are known in the art, e.g., as described in O'Farrell,
J. Biol. Chem.
250, 4007-4021 (1975) and O'Farrell et al.,
Cell,
12:1133-1142 (1977), the entirety of which are incorporated herein by reference. Gels cannot be used for the determination of absolute isoelectric points, but rather to an observed value due to the fact that the running conditions, e.g., high urea concentration, are not ideal (in the physical-chemistry meaning of the word—the pKa values of the side chains are rather different between water and high concentrations of urea). NEPHGE gels cannot be used for the determination of absolute isoelectric points of proteins. The isoelectric point of a protein is usually determined in reference to a stable pH gradient formed during isoelectric focusing. As discussed in O'Farrell (1977), the best resolution of acidic proteins is obtained with equilibrium IEF since the region of the gels containing acidic proteins is compressed in NEPHGE. The best resolution of basic proteins is with a pH 7-10 NEPHGE gel. For the highest resolution of the entire range of proteins, two gels are preferably used: (1) an IEF gel for acidic proteins; and (2) a NEPHGE gel for basic proteins. Of course, the precise image obtained depends upon many factors including, but not limited to, chemicals such as ampholytes, physical parameters such as gel dimensions and/or temperature and/or the electrical parameters employed.
Once a 2DGE gel is run, the image can be revealed by a number of ways including: staining (e.g., with Coomassie blue, silver or fluorescent or immunological staining); direct measurement of the radioactivity (using devices that can detect the radioactivity (including so-called &bgr;-imagers, or phosphor image technologies); or photographic film sensitive to the radioactivity); fluorescent enhancement of the radiographic emissions (using various fluorophores) or combinations of the above. In addition, samples can be treated before electrophoresis (e.g., with monobromobimane and related compounds) so that the proteins are detectable after electrophoresis using some of the above methods. For example, after electrophoresis, a 2DGE gel can be fixed with methanol and acetic acid, treated with AMPLIFY® (Amersham), and dried. The gel is then placed in contact with X-ray film and exposed. The gel can be exposed for multiple time periods to compensate for the lack of dynamic range of X-ray films. Each film image comprises a multiplicity of “spots” of differing position, size, shape, and/or optical density. The spots on the image are analyzed to determine the correspondence between spots and proteins.
Manual visual inspection and analysis of gel images can be done under controlled conditions and within specific ranges (Jungblut et al., Quantitative analysis of two-dimensional electrophoretic protein patterns: Comparison of visual evaluation with computer-assisted evaluation. In: Neuhoff, V. (Ed.) Electrophoresis '84, Verlag Chemie GmbH, Weinheim, 301-303 and Andersen et al.,
Diabetes,
Vol. 44, April 1995, 400-407). Analysis of one film can take in excess of eight hours, even for one having significant skill and experience in this art. Typically, certain methods of obtaining images of the gels are non-linear (e.g., silver staining and/or X-ray film) and it is often necessary to make corrections for this if the full range of protein expression is to be covered (this can be up to a ratio of 1:100,000 for the protein of lowest to highest expression). Further, quantification with visual analysis is limited. Typically, visual analysis only detects changes in protein amounts of a factor greater than or equal to 2.
Various computer programs and computer evaluation systems have been developed to improve quantification and assist in evaluation of gel films, e.g., PDQUEST (Protein Database Inc., New York), BioImage (Millipore, Bedford, Mass.), Phoretix (Phoretix International, Newcastle, UK), and Kepler (Large Scale Biology Corporation, Rockville, Md.). To use a computer program such as BioImage (BioImage, Ann Arbor, Mich.), the image of the gel on the film is scanned into a computer. The digitized gel image is analyzed by the computer program. Each spot is assigned an intensity value, such as an integrated optical density percentage (IOD %), and a position on the g
Fey Stephen J.
Larsen Peter Mose
Azarian Seyed
Patel Jayanti K.
Sterne Kessler Goldstein & Fox P.L.L.C.
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