Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-02-25
2004-01-13
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C436S173000, C436S175000, C436S086000, C436S087000, C436S089000
Reexamination Certificate
active
06677114
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of protein separation and proteomics.
BACKGROUND OF THE INVENTION
A goal of genomics research and differential gene expression analysis is to develop correlations between gene expression and particular cellular states (e.g., disease states, particular developmental stages, states resulting from exposure to certain environmental stimuli and states associated with therapeutic treatments). Such correlations have the potential to provide significant insight into the mechanism of disease, cellular development and differentiation, as well as in the identification of new therapeutics, drug targets, and disease markers. Correlations of patterns of gene expression can also be used to provide similar insights into disease and organism metabolism that can be used to speed the development of agricultural products, transgenic species, and for metabolic engineering of organisms to increase bioproduct yields or desirable metabolic activities.
Many functional genomic studies focus on changes in mRNA levels as being indicative of a cellular response to a particular condition or state. Recent research, however, has demonstrated that often there is a poor correlation between gene expression as measured by mRNA levels and actual active gene product formed (i.e., protein encoded by the mRNA). [4] This finding is not surprising since many factors—including differences in translational efficiency, turnover rates, extracellular expression or compartmentalization, and post-translational modification—affect protein levels independently of transcriptional controls. Thus, the evidence indicates that functional genomics is best accomplished by measuring actual protein levels (i.e., utilizing proteomic methods) rather than with nucleic acid based methods. The successful use of proteins for functional genomic analyses, however, requires reproducible quantification and identification of individual proteins expressed in cell or tissue samples.
It is at the protein level that metabolic control is exercised in cells and tissues. Comparison of the levels of protein expression between healthy and diseased tissues, or between pathogenic and nonpathogenic microbial strains, can speed the discovery and development of new drug compounds or agricultural products. Analysis of the protein expression pattern in diseased tissues or in tissues excised from organisms undergoing treatment can also serve as diagnostics of disease states or the efficacy of treatment strategies, as well as provide prognostic information regarding suitable treatment modalities and therapeutic options for individual patients.
Many proteins are expressed at varying levels in different cells. Proteins extracted from tissue or cell samples, using conventional techniques, must first be separated into individual proteins by gel or capillary electrophoresis or affinity techniques, before the individual proteins levels can be compared both within a sample and across samples obtained from different tissue sources. Because of the number of proteins expressed by a cell at any given time, multiple electrophoretic techniques (e.g., isoelectric focussing followed by electroporation through a polyacrylamide gel) are often applied to isolate all the individual proteins contained in a given sample.
Several techniques have been used to quantify the relative amounts of each protein present after the separation, including: staining proteins separated in a polyacrylamide gel with dyes (e.g., Brilliant Blue and Fast Green), with colloidial metals (e.g., gold or silver staining), or by prior labelling of the proteins during cellular synthesis by the addition of radioactive compounds (e.g., with 35S-methionine or 14C-amino acids, or 3H-leucine). Staining techniques yield poorly quantitative results because varying amounts of stain are incorporated into each protein and the stained protein must be resolved against the stained background of the gel or electroblotting substrate. Since radioactive labels are applied only to the proteins prior to separation, they overcome the background problem of staining techniques. However, feeding radioactive compounds to human subjects or handling radioactive materials in an uncontrolled field environment (e.g., crop plants) restricts the usefulness of this approach. Both staining and radiolabelling techniques also require inordinately long times to achieve detection. Staining and destaining of gels is a diffusion limited process requiring hours. Radiolabels must be quantified by exposing the labelled gel to photographic film or a phosphor screen for several hours to days while waiting for the radioactive decay process to produce a quantitative image. Direct infrared spectrophotometric interrogation of the proteins in a gel has also been used previously as a method for providing quantitative protein expression data. However, this quantitative resolution possible from this approach is adversely affected by variations in gel thickness and differential spreading of the protein spot between gels (changing the local concentration). Furthermore, the comparatively low absorption cross-section of proteins in the infrared limits the detection sensitivity. Analysis of the protein expression pattern does not provide sufficient information for many applications.
Several methods have also been proposed for the identification of proteins once they are resolved. The most common methods involve referencing the separation coordinates of individual proteins (e.g., isoelectric point and apparent molecular weight) to those obtained from archived separation coordinate data (e.g., annotated 2-D gel image databases) or control samples, performing a chemilytic or enzymatic digestion of a protein coupled with determination of the mass of the resulting peptide fragments and correlating this peptide mass fingerprint with that predicted to arise from the predicted genetic sequence of a set of known proteins (see James, P., M. Quandroni, E. Carafoli, and G. Gonnet,
Biochem. Biophys. Res. Commun.,
195:58-64 (1993); Yates, J. R., S. Speicher, P. R. Griffin, and T. Hunkapiller,
Anal. Biochem.,
214:397-408 (1993)), the generation of a partial protein sequence that is compared to the predicted sequences obtained from a genomic database (see Mann, M., paper presented at the IBC Proteomics conference, Boston, Mass. (Nov. 10-11, 1997); Wilm, M., A. Shevchenko, T. Houthaeve, S. Breit, L. Schweiger, T. Fotsis and M. Mann,
Nature,
379:466-469 (1996); Chait, B. T, R. Wang, R. C. Beavis and S. B. H. Kent,
Science,
262:89-92 (1993)), or combinations of these methods (see Mann, M., paper presented at the IBC Proteomics conference, Boston, Mass. (Nov 10-11, 1997); Wilm, M., A. Shevchenko, T. Houthaeve, S. Breit, L. Schweiger, T. Fotsis and M. Mann,
Nature,
379:466-469 (1996); Chait, B. T, R. Wang, R. C. Beavis and S. B. H. Kent,
Science,
262:89-92 (1993)). Recent work indicates that proteins can only be unambiguously identified through the determination of a partial sequence, called a protein sequence tag (PST), that allows reference to the theoretical sequences determined from genomic databases (see Clauser, K. R., S. C. Hall, D. M. Smith, J. W. Webb, L. E. Andrews, H. M. Tran, L. B. Epstein, and A. L. Burlingame, “
Proc. Natl. Acad. Sci.
(
USA
), 92:5072-5076 (1995); Li, G., M. Walthan, N. L. Anderson, E. Unworth, A. Treston and J. N. Weinstein,
Electrophoresis,
18:391-402 (1997)). However, between 8 to 18 hours is currently required to generate a PST for a single protein sample by conventional techniques, with a substantial fraction of this time devoted to recovery of the protein sample from the separation method in a form suitable for subsequent sequencing (see Shevchenko, A., et al.,
Proc. Natl. Acad. Sci.
(
USA
), 93:14440-14445 (1996); Mark, J., paper presented at the PE/Sciex Seminar Series, Protein Characterization and Proteomics: Automated high throughput technologies for drug discovery, Foster City, Calif. (March, 1998). This makes the identification of all separated proteins from a
Hall Michael P.
Peterson Jeffrey N.
Petesch Robert
Schneider Luke V.
Horlick Kenneth R.
Kim Young
Target Discovery, Inc.
Townsend and Townsend / and Crew LLP
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