High performance multidimensional proteome analyzer

Details High performance multidimensional proteome analyzer High performance multidimensional proteome analyzer

1999-04-23

2001-08-21

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S456000, C204S459000, C204S466000, C204S467000, C204S606000, C204S610000, C204S612000, C204S616000, C204S618000

Reexamination Certificate

active

06277259

ABSTRACT:

BACKGROUND OF THE INVENTION
Today, most clinical laboratory tests are individual tests that analyze and quantify the level of a specific protein in a clinical sample such as a bodily fluid proteome (BFP). The effectiveness of proteome analysis is dependent upon the electrophoretic separation technology utilized. Most commonly, one-dimensional eletrophoretic separation methods, such as polyacrylamide gel electrophoresis, are utilized. One-dimensional separation typically resolves about 100 distinct zones per gel.
The advent of the Age of Genomics, however, has increased the perception of what is required in the post-genome era. DNA sequencing information alone cannot accurately predict many critical events. These include whether and when gene products are translated, the relative concentration of gene products, the extent of post-translational modifications of the gene products, and the effects of under- or over-expression. In cellular systems, particularly those subject to changes in physiological condition or disease, dynamic genome activity, such as up- and down-regulation, can cause the appearance and disappearance of hundreds or thousands of spots per electrophoretic image. The changes reflected in the electrophoretic image must be evaluated and analyzed quantitatively by a high performance image-processing algorithm. To adequately decipher multigenic phenomena and interactions between genes, the sensitivity of the analysis system should permit simultaneous, quantitative tracking of 40-70% of all genome activity.
Two- or multi-dimensional separation methods allow specific recognition of nearly all detectable protein spots in a BFP. Multi-dimensional electric field mediated analysis yields resolutions of up to tens of thousands of distinct zones per gel. Hence, multi-dimensional separation is far superior to one-dimensional separation for separation of complex mixtures of protein molecules and for tracking multigenic phenomena at the cell, tissue and even organism level.
Although two-dimensional separation technology is twenty years old, employment of the technology has been hindered by a number of obstacles. These include an inability to produce consistent results from the same samples, and a lack of an adequate method for analysis of the plethora of information generated by multi-dimensional analysis. Often, analysis of a two-dimensional separation pattern consists simply of holding two gels up to a light and, unless differences are visible in the peripheries of the gels, disposing of the bulk information the gels represent. Another major obstacle, relating to the lack of an adequate analysis method, is the absence of a simple system to identify and quantify individual proteins or groups of proteins. Moreover, despite numerous refinements in electrophoretic techniques over the past decade, two-dimensional separation is still tedious and inefficient. The time required to prepare, load, separate and visualize complex mixtures of protein molecules is substantial. This is especially problematic, since throughput is the single most important factor influencing the cost effectiveness of proteome analysis. Though automated laboratory analyzers simplify these complex tasks with robotics, robotics are not cost effective in small outpatient health care units.
Despite these problems, electric field mediated two-dimensional separation methods, such as two-dimensional gel electrophoresis, continue to gain importance in biological research and direct clinical applications. One reason is the perceived potential to identify certain protein molecules or groups of proteins that are up- or down regulated, and whose concentration can be correlated with a disease and ration varies with the disease progress.
Conventional two-dimensional gel electrophoresis-based protein separation methods comprise two separation dimensions: isoelectric focusing, (“IEF”) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (“SDS-PAGE”). IEF is almost exclusively the first separation dimension. In IEF, amphoteric molecules such as proteins are separated by electrophoresis in a pH gradient generated between a cathode and an anode. IEF takes advantage of the fact that each protein has a characteristic pH at which it is electrically neutral. This characteristic pH is the isoelectric point (pI) of the protein. Under the influence of an electric field, charged sample components migrate through an electrophoresis medium (a solution or a gel). If a sample component has a net negative charge, it migrates towards the anode. During migration, the negatively charged sample encounters a progressively lower pH, thus becoming more positively charged. Eventually, the pI is reached where the net charge of the sample component is zero. At the pI, migration stops and the sample component is “focused” in a tight zone. Likewise, if a sample component is positively charged, it will migrate towards the cathode. In this manner, each sample component migrates to its isoelectric point. IEF is a true electric field mediated focusing technique since protein molecules that diffuse out of the focused zone acquire charge and are pulled back into the zone where the net charge is zero.
The pH gradient, which is key to the success of IEF, is provided by molecules called “carrier ampholites”. Carrier ampholites are polyamino-polycarboxic acids having gradually differing pI values. Ampholite mixtures are available in various narrow and broad pH ranges. Typically, an anti-convective media such as polyacrylamide or agarose is used. It is also possible to immobilize pH gradients on a suitable matrix such as polyacrylamide or ampholite strips. With immobilized pH gradients, IEF routinely provides a resolution of 0.1 to 0.01 pI units.
Relatively high electric field strengths are necessary to obtain rapid isoelectric focusing. Use of capillary dimensions (i.e. dimensions less than 0.2 mm I.D.) provides efficient dissipation of Joule heat and permits the use of such high field strengths. In capillary dimensions, IEF separations can be carried out in free solution or in entangled polymer networks.
As noted above, the second separation dimension is typically carried out by SDS-PAGE. SDS-PAGE involves complex relationships among several factors. These factors include separation length, gel composition, gel pore size, electric field strength, ionic moiety, buffer composition and the mode of migration of the polyion through the gel matrix. In conventional SDS-PAGE separations, biopolymers migrate under the influence of an electric field by tumbling through pores whose average radii are much larger that the radius of gyration of the analyte. Migrating samples are thereby size-ordered based on the time required to find a path through the pores of the gel matrix. This type of migration is known as separation in the Ogston regime, and is usually quite time-consuming. Larger molecules, i.e. those molecules whose radii of gyration are larger than the average pore size, are impeded and become oriented towards the electric field while migrating through the pores. This process, which is called reptation, can be induced through increases in either the gel concentration or the applied electric field strength.
The use of increased electric field strengths (typically greater than 100V/cm) necessitates thickness reduction in planar systems. Thickness reduction enhances the ability to dissipate heat and thereby reduces the effects of Joule heat. Some emerging capillary electrophoresis methods employ narrow-bore capillary columns having large surface-to-volume ratios to effectively dissipate heat. In planar electrophoretic systems, the surface-to-volume ratio is increased through thickness reduction, ideally converging towards capillary dimensions. This is known as “ultra-thin” gel electrophoresis. Rapid biopolymer separation, for example, requires gel-filled separation platforms having a thickness of no more than 0.25 mm. The use of 0.1 mm thick gels for biopolymer separation allows as much as a five-fold increase in electric field strength. Use of polyacrylamide

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