Optics: measuring and testing – By electrophoresis
Reexamination Certificate
2002-01-17
2004-11-09
Mathews, Alan (Department: 2851)
Optics: measuring and testing
By electrophoresis
C204S456000
Reexamination Certificate
active
06816259
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the analysis and separation of biomolecules. More particularly, the present invention relates to a method and apparatus for the automated excision of individual protein samples from two-dimensional electrophoresis gels for subsequent analysis of protein content.
BACKGROUND OF THE INVENTION
The method and apparatus described herein are used for the automated excision of individual samples from two-dimensional (“2D”) electrophoresis gels for subsequent analysis (referred to herein as the “Invention”). The Invention may be used in any art or occupation where the user wishes to separate and analyze proteins or other substances that are identifiable by 2D gel electrophoresis techniques, or any other technique that results in the physical separation of substances within planar and cuttable materials.
By way of example, one such art is “proteomics,” especially in conjunction with a related art, “genomics.” Proteomics is the study of the protein complement that an organism is capable of producing, whereas genomics is the study of deoxyribonucleic acid (“DNA”), its genes, and the processes that lead to the creation of proteins. Proteomics provides data on the outcome of gene expression. Genomics provides the comprehensive gene sequence data, often derived by microarray analysis, required to advance protein research.
In complex organisms, individual cells may selectively express genes in their DNA to yield sets of proteins required for specific cell or organ functions. Much current scientific effort is directed to creating databases concerning how these genes are regulated and how this regulation may change in disease or other states, whether before and after treatment.
In order to evaluate the effects of gene regulation, methods must be used that measure, separate, and qualitatively and quantitatively analyze proteins, which are one output of gene expression. One currently favored protcomic technique is 2D polyacrylamide gel electrophoresis. This technique separates complex mixtures of proteins so that they can be isolated, quantified, identified and then assessed for their role in a disease process or as a target for novel drugs.
One approach to proteomic study using 2D gel techniques can be considered as comprising eight individual operations (see FIG.
1
):
1. Solubilization
16
—The proteins in a sample 15 of cells or tissue are released from the underlying cellular or tissue matrix by solubilizing the proteins with detergents.
2. Separation
17
—The solubilized proteins are then physically separated into a square gel array using 2D gel electrophoresis.
3. Staining
18
—The separated proteins are demonstrated in the gel by staining with or attaching Coomassie brilliant blue, silver staining, SYPRO ruby, fluorescent compounds, or by other appropriate techniques.
4. Imaging
19
—The stained 2D gels are imaged by electronic optical or other means for resolving protein sample spots which are potentially interesting. For example, proteins that occur differentially in diseased but not healthy tissue could be considered of interest.
5. Picking
20
—The spots of gel containing the proteins of interest are excised from the main gel matrix.
6. Digestion of protein into peptides
21
—The proteins are broken down, usually enzymatically, into constituent peptides whose masses can be measured by mass spectrometry.
7. Mass spectral analysis
22
—The size of the isolated and digested protein peptides are measured using a matrix assisted laser desorption ionization-time of flight (“MALDI-TOF”) mass spectrometer, or analyzed by liquid chromatography-mass spectrometry, quadropole time of flight, or other means.
8. Identification
23
,
24
—The proteins are identified by matching the masses of the set of peptide fragments to fragments predicted by public and private databases after similar proteolytic (enzymatic) treatment. Once identified, the role of each protein in a disease process or as a potential point of intervention in a disease process (e.g., a drug target) can be considered along with information from pathology, pharmacology and known biological pathways.
In conjunction with computer databases and analysis, 2D gel electrophoresis can provide a means to physically resolve the proteome of a tested sample according to each protein's isoelectric point, reflected on one axis of the planar 2D gel sample, and its molecular weight or size, reflected by a corresponding perpendicular planar axis. Thus, 2D gel analysis of any given sample may produce a “fingerprint” that reflects an orthogonal planar distribution of its protein complement according to individual protein characteristics. Once prepared, resolved 2D gels may be translated by staining, imaging, and bioinformatic software into high-resolution digital protein maps, which may be stored for future use in computer or other databases. The resulting data may be used to determine the protein profiles of different tissues in both healthy and disease states, and ultimately for proteome libraries.
In addition, individual proteins may be excised from 2D gels, split into peptide fragments, and measured using mass spectrometry or other means. However, the large-scale study of proteins and protein networks is currently limited in part by the ability to physically isolate, segregate, and study individual proteins. Currently operations like those in
FIG. 1
are done in a sequential and modular fashion. The output of each step is transferred manually from operation to operation. These individual unconnected manual operations make the technique slow and cumbersome, prone to error due to the repetitive nature of each manual step, and subject to contamination, for example, by keratin contamination from skin during handling.
Scientists studying proteomics and genomics, and others, are extremely interested in rapid, accurate high throughput methods and instruments to carry out protein analysis. It is clear that advances in robotics and software/computing technology could improve the throughput and rate of the analysis.
One U.S. company, BioRad Laboratories, is developing a protein-picking system in collaboration with a company called AARM (an Australian firm). However, among other distinctions, their system is only semi-automated, and the user must manually identify the proteins to be picked from a particular 2D gel. Furthermore, the BioRad system does not use information stored in 2D gel databases to identify proteins of interest to be excised. Finally, the BioRad system does not have the capability of utilizing excision tools of different sizes based upon the size of the protein in the 2D gel.
Although there is other information to suggest other interest in the field, see e.g., Anderson, et at., U.S. Pat. No. 5,993,627 at Columns 26-28, there appears to be no claimed invention or art providing the novel elements, means and utility of the claimed Invention.
SUMMARY OF THE INVENTION
The Invention offers a method and automated apparatus for the separation, excision, and high throughput handling of protein samples demonstrated via 2D gel for further analysis. The Invention utilizes a laboratory-grade XYZ Gantry robot, a novel approach to the identification of the proteins of interest to be excised, novel tools for the excision of the protein samples from the 2D gels, and novel means for controlling robot and process steps to accomplish selective and automated protein sample excision.
Currently, the process of protein excision is performed by hand, is extremely labor-intensive, and is prone to error. The manual process is also susceptible to contamination, rendering the protein under analysis virtually useless. The use of the laboratory robot and the novel excision tools described herein will increase the efficiency of protein excision and will greatly reduce contamination by minimizing user handling of the protein samples.
REFERENCES:
patent: 4684244 (1987-08-01), Butts et al.
patent: 4909920 (1990-03-01), Sarrine et al.
patent: 6064754 (2000-05-01), Parekh et al.
patent: WO 98/23950 (1998-06-01), N
Auton Kevin
Byatt David
Ryan Paul Thomas
Mathews Alan
Rader & Fishman & Grauer, PLLC
LandOfFree
Method and apparatus for automated excision of samples from... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and apparatus for automated excision of samples from..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for automated excision of samples from... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3286206