Protein separation via multidimensional electrophoresis

Details Protein separation via multidimensional electrophoresis Protein separation via multidimensional electrophoresis

2003-01-13

2004-11-16

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S451000, C204S601000, C435S004000, C435S006120, C436S173000, C436S175000, C436S086000, C436S087000, C436S089000

Reexamination Certificate

active

06818112

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/or 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). 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 of individual proteins expressed in cell or tissue samples.
Two-dimensional (2-D) gel electrophoresis is currently the most widely adopted method for separating individual proteins isolated from cell or tissue samples [5, 6, 7]. Evidence for this is seen in the proliferation (more than 20) of protein gel image databases, such as the Protein-Disease Database maintained by the NIH [8]. These databases provide images of reference 2-D gels to assist in the identification of proteins in gels prepared from various tissues.
Capillary electrophoresis (CE) is a different type of electrophoresis, and involves resolving components in a mixture within a capillary to which an electric field is applied. The capillary used to conduct electrophoresis is filled with an electrolyte and a sample introduced into one end of the capillary using various methods such as hydrodynamic pressure, electroosmotically-induced flow, and electrokinetic transport. The ends of the capillary are then placed in contact with an anode solution and a cathode solution and a voltage applied across the capillary. Positively charged ions are attracted towards the cathode, whereas negatively charged ions are attracted to the anode. Species with the highest mobility travel the fastest through the capillary matrix. However, the order of elution of each species, and even from which end of the capillary a species elutes, depends on its apparent mobility. Apparent mobility is the sum of a species electrophoretic mobility in the electrophoretic matrix and the mobility of the electrophoretic matrix itself relative to the capillary. The electrophoretic matrix may be mobilized by hydrodynamic pressure gradients across the capillary or by electroosmotically-induced flow (electroosmotic flow).
A number of different electrophoretic methods exist. Capillary isoelectric focusing (CIEF) involves separating analytes (such as proteins) within a pH gradient according to the isoelectric point (i.e., the pH at which the analyte has no net charge) of the analytes. A second method, capillary zone electrophoresis (CZE) fractionates analytes on the basis of their intrinsic charge-to-mass ratio. Capillary gel electrophoresis (CGE) is designed to separate proteins according to their molecular weight. (For reviews of electrophoresis generally, and CIEF and CZE specifically, see, e.g., Palmieri, R. and Nolan, J. A., “Protein Capillary Electrophoresis: Theoretical and Experimental Considerations for Methods Development,” in
CRC Handbook of Capillary Electrophoresis: A Practical Approach
, CRC Press, chapter 13, pp. 325-368 (1994); Kilar, F., “Isoelectric Focusing in Capillaries,” in
CRC Handbook of Capillary Electrophoresis: A Practical Approach
, CRC Press, chapter 4, pp. 95-109 (1994); and McCormick, R. M., “Capillary Zone Electrophoresis of Peptides,” in
CRC Handbook of Capillary Electrophoresis: A Practical Approach
, CRC Press, chapter 12, pp. 287-323 (1994). All of these references are incorporated by reference in their entirety for all purposes).
While 2-D gel electrophoresis is widely practiced, several limitations restrict its utility in functional genomics research. First, because 2-D gels are limited to spatial resolution, it is difficult to resolve the large number of proteins that are expressed in the average cell (1000 to 10,000 proteins). High abundance proteins can distort carrier ampholyte gradients in capillary isoelectric focusing electrophoresis and result in crowding in the gel matrix of size sieving electrophoretic methods (e.g., the second dimension of 2-D gel electrophoresis and CGE), thus causing irreproducibility in the spatial pattern of resolved proteins [20, 21 and 22]. High abundance proteins can also precipitate in a gel and cause streaking of fractionated proteins [20]. Variations in the crosslinking density and electric field strength in cast gels can further distort the spatial pattern of resolved proteins [23, 24]. Another problem is the inability to resolve low abundance proteins neighboring high abundance proteins in a gel because of the high staining background and limited dynamic range of gel staining and imaging techniques [25, 22]. Limitations with staining also make it difficult to obtain reproducible and quantifiable protein concentration values, with average standard variations in relative protein abundance between replicate 2-D gels reported to be 20% and as high as 45% [4]. In some recent experiments, for example, investigators were only able to match 62% of the spots formed on 3-7 gels run under similar conditions [21; see also 28, 29]. Additionally, many proteins are not soluble in buffers compatible with acrylamide gels, or fail to enter the gel efficiently because of their high molecular weight [26, 27].
SUMMARY OF THE INVENTION
The present invention provides a variety of electrophoretic methods and apparatus for separating mixtures of proteins. The methods involve conducting multiple capillary electrophoresis methods in series, wherein samples for each method other than the initial method contain only a subset of the proteins from the preceding step (e.g., from fractions containing resolved protein from the preceding method). By using a variety of techniques to control elution during electrophoresis, the methods are capable of resolving proteins in even complex mixtures such as obtained from tissues and native cells. Utilizing various labeling schemes and detection methods, certain methods can provide quantitative information on the amount of each of the separated proteins. Such information can be used in the development of protein databases in which proteins expressed under certain conditions are characterized and catalogued. Comparative studies to identify proteins that are differentially expressed between different types of cells or tissues can also be conducted with the methods of the present invention. The methods can also be used in diagnostic, structure activity and metabolic engineering studies.
In general, the methods involve performing a plurality of electrophoretic methods in series. Each method in the series i

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