Apparatus and methods for high-resolution and high-sensitivity assays

Abstract

An apparatus allows separation, fractionation isolation and fraction collection simultaneously. The device consists of two major pieces, with one piece slides relatively to the other to facilitate the switching between separation and fractionation.

Description

This is a continuation of U.S. application Ser. No. 10/371,981 filed on Feb. 21, 2003, now U.S. Pat. No. 7,189,370 which claims the priority of the following pending applications: U.S. Provisional Patent Application Ser. No. 60/359,391, filed on Feb. 22, 2002; U.S. Provisional Patent Application Ser. No. 60/383,190 filed on May 3, 2002; and U.S. Utility patent application Ser. No. 10/076,012 filed on Feb. 11, 2002.

FIELD OF THE INVENTION

The present invention relates generally to the field of sensitive detection of molecules. More particularly, the present invention relates to methods and apparatus of use in multi separation techniques to sensitively measure the quantities of extracts of herbs, plants, organisms/tissues/biological fluids and other natural materials, and protein/peptide samples, using a novel apparatus that integrates one analytical technique to another analytical technique.

DESCRIPTION OF RELATED ARTS

The technology advancement has greatly facilitated the separation and identification of proteins in complex protein mixtures. The most popular technologies include the two-dimensional gel electrophoresis (2DE) followed by mass spectrometry (MS) detection, the difference gel electrophoresis (DIGE), the shotgun approach, the isotope-coded affinity tag (ICAT) technique, etc. A common argument in favor of 2DE is that a comparison can readily be made between two gels and thus proteome differences can be detected. However, 2DE is still problematic because of the gel-to-gel irreproducibility, varying staining efficiency of individual gels, and bias against some protein classes such as membrane proteins. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy numbers is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. All these problems are more or less resulted from the inadequate LOD and dynamic range of the analytical tools that are being used. Considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understanding biological changes, and 2DE remains to be one of the main techniques that can offer sufficient resolution to address this issue at a functional level. In this project, we will push the limits (e.g. LOD and dynamic range) of 2DE for LAP profiling. The following section presents an overview of 2DE and the related technologies.

2DE. The first 2-dimensional (2D) separations can be attributed to the work of Smithies and Paulik who used a combination of paper and starch gel electrophoresis for the separation of serum proteins. Subsequent developments in electrophoretic technology, such as the use of polyacrylamide as a support medium and the use of polyacrylamide concentration gradients, were rapidly applied to 2D separations. In particular, the application of isoelectric focusing (IEF) techniques developed by O'Farrell to 2D separations made it possible for the 1^st-D separation to be based on the charge properties of the proteins. The coupling of IEF with SDS-PAGE in the 2^nd-D resulted in a 2DE method that separated proteins according to two independent parameters, isoelectric point and molecular weight. This methodology was then adapted to a wide range of samples with differing solubility properties by the use of urea and applied to the analysis of protein mixtures of whole cells and tissues.

There are three basic methods for the 1^st-D, ISO-Dalt, IPG-Dalt, and NEpHGE. In ISO-Dalt, the pH gradient is formed by carrier ampholytes during the separation. At equilibrium, each protein is focused to a position corresponding to its isoelectric point. The disadvantage of this technique is the lot-to-lot variability of the carrier ampholytes. ISO-Dalt is still used because the gels can be prepared easily without having to make sophisticated gradients. IPG-Dalt uses an immobilized ampholyte strip to create a pH gradient. This technique provides better batch-to-batch reproducibility because the strips are commercially available in a dry state that can be easily hydrated. However, problems have been reported, such as loss of large proteins in the 1^st-D, unsatisfactory resolubilization in the 2^nd-D, and precipitation by unpolymerized immobilines. Non-equilibrium pH gradient electrophoresis (NEpHGE) is typically used to resolve extremely basic proteins. It differs from the other two in that: (i) the polarity of the power supply is reversed at a predetermined volt-hour value during the separation; and (ii) the focusing process is stopped before reaching equilibrium.

After the 1^st-D separation, the buffer in the gel is exchanged with SDS buffer. The partially resolved proteins from the 1^st-D are then transferred to a slab-gel for SDS-PAGE, the 2^nd-D separation. At the end of the 2^nd-D separation, the separated proteins are stained and detected. The LOD and dynamic range depend on the stain method that is used.

Common stain methods. Coomassie Brilliant Blue is often utilized to stain proteins in SDS-PAGE gels for both qualitative and quantitative detections. The advantages of Coomassie staining methods include quantitative binding of dye to proteins, low price, and good reproducibility. Usually, it is compatible with MS analysis. When a protein is detectable with Coomassie Brilliant Blue, as a rule of thump, enough protein is present for appropriate mass spectrometry analysis with MALDI-TOF. The disadvantages are the long staining times, relatively low sensitivity, and narrow dynamic range.

Silver Staining is another popular staining method employed for protein detection. In this method, the gel is preserved with soluble silver ions and developed by treatment with formaldehyde, which reduces silver ions to form an insoluble brown precipitate of metallic silver. This reduction is promoted by protein. It is a sensitive (sub-nanogram detection limit) method for permanent staining of proteins in SDS-PAGE gels. However, it is incompatible with MS, and has a narrow dynamic range.

Fluorescent dye staining is another technology that is widely used for proteome analysis due to its advantages of high sensitivity combined with wide linear dynamic range. For example, SYPRO Ruby Staining has a sensitivity of ˜1 ng per spot and a linear dynamic range of ˜10³. These numbers reflect the highest level of state-of-the-art technology, but higher sensitivities and wider dynamic ranges are still greatly demanded.

Electroblotting of proteins from 2DE. Because of the ability of 2DE to separate simultaneously up to several thousand proteins using large-format gels, it has become the method of choice for the analysis of protein expression in complex biological systems. A variety of methods are available now to further identify and characterize proteins separated by 2DE. Many of these methods depend on the technique of Western electroblotting in which proteins separated by 2DE are transferred (“blotted”) by the application of an electric field perpendicular to the plane of the gel onto the surface of an inert membrane, such as nitrocellulose.

Two types of apparatus are in routine use for electroblotting. In the first approach (known as “tank” blotting), a sandwich assembly of gel and blotting membrane is placed vertically between two platinum wire electrodes arrays contained in a tank filled with a blotting buffer.¹⁹ In the second type of procedure (know as “semidry” blotting), the gel-blotting membrane assembly is sandwiched between two horizontal plate electrodes, typically made of graphite.

Proteins immobilized in this way are readily accessible to interaction with probes, such as polyclonal antibodies and monoclonal antibodies or other ligands specific for the proteins being analyzed. This approach has been used extensively for specific (known) protein detection and quantitation. Recently, 2DE is used for micro-preparative purification of proteins for subsequent chemical characterization, which has often been applied for proteomic research.²¹ In this approach the protein, while still on the surface of the inert membrane support, can be analyzed by numerous characterization techniques, including N-terminal and internal amino acid sequencing, amino acid compositional analysis, peptide profiling, and mass spectrometry.

Capillary SDS-PAGE. Capillary SDS-PAGE is a miniaturized gel electrophoresis platform that has many advantages over traditional slab-gel techniques (e.g. higher separation efficiency, shorter separation time, lower mass detection limit, more convenient to implement automated operation, etc.). Capillary SDS-PAGE is a special type of CGE, but for the simplicity of expression in the text of this proposal we treat it as CGE. Typically, the separated proteins in CGE are detected in-column by either an Ultraviolet (UV) absorbance or a laser-induced fluorescence (LIF).

UV absorption is arguably the most frequently used detection mode in CE. It is also commonly employed in CGE since protein-SDS complexes absorbs light around 280 nm due to the aromatic side groups of amino acids and around 200˜220 nm due to the peptide bonds between amino acids. LIF detection is preferred when a low limit of detection and a wide dynamic range are desired. Native fluorescence of proteins has been explored for direct protein detection, but it is not widely accepted because of the use of expensive UV lasers. Often, proteins are somehow fluorescently labeled, and then measured by a LIF detection system using a relatively inexpensive laser such as an air-cooled argon ion or helium-neon laser.

Labeling proteins reliably and reproducibly is challenging, although much progress has been made. Proteins have been covalently linked with fluorescent dyes mainly via the amine groups on the proteins. These proteins can then be bound with SDS and separated by CGE. Protein concentrations of as low as 3×10⁻¹⁰ M (4˜10 ng/mL) were successfully analyzed by CGE. However, most of these protocols are cumbersome and suffer from incomplete and ambiguous labeling, resulting in complex electropherograms. Alternatively, proteins can react with SDS first and the protein-SDS complexes are then dynamically labeled with fluorescent dyes before electrophoresis. Presumably due to the low binding efficiency and high background noise, only moderate low LODs (30˜500 ng/mL) were achieved.

This invention describes a method and the associated instrument to map the LAPs in complex biological samples. To reduce the HAP interference with the detection of the LAPs, we deplete the HAPs from the biofluids using affinity depletion techniques. The IEF fractionation device can separate/concentrate/fractionate the LAPs, and all fractions can be further separated by parallel CGE. The method and apparatus can achieve a LOD of ≦10×10⁻¹⁵ mol/mL, a dynamic range of ≧10⁵ and a resolving power of ≧5,000 proteins for LAP mapping and profiling.

SUMMARY OF THE INVENTION

The present invention solves a long-standing need in the art by providing a high-resolution fractionation device for high resolution and high sensitivity assays of protein and peptide samples. The apparatus can be used for variety of applications, including extracts of herbs, plants, organisms/tissues/biofluids and other natural materials, microorganisms, DNA, RNA, carbohydrates, polysaccharides and lipids.

In one aspect of the present invention, the fractionation device incorporates a separation scheme, such as IEF. The pre-separated fractions are isolated locally to maintain the resolution of the above said separation.

In one embodiment, a pre-separated sample (e.g. from an HPLC or a CE instrument) is first introduced into the fractionation device. The fractionation device then segments and isolates the said sample locally before fractionation.

In one particular embodiment, the integrated device has 2 pieces. The top piece moves relatively to the bottom piece to facilitate the pre-separated sample bands to be transferred to fraction collection containers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the fractionation device

FIG. 2. Schematic design of the fractionation device assembly

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fractionation device for high resolution and high sensitivity assays of complex biological samples, such as herb extracts and protein/peptides samples from proteomic field. FIG. 1 presents a schematic diagram of the fractionation device. Because it operates like a valve, it is sometimes called a valve device. The device consists of two pieces (X and Y) and works as a sliding valve. The thin and thick lines represent capillary tubes. Pieces X and Y can be made of materials such as PEEK (polyetherether ketone), nylon, PVC, alumina, ceramic and silica. Holes will be drilled on X and Y strips to host the capillaries. Adhesives can be applied to secure the capillaries in position. When pieces X and Y are aligned as depicted in FIG. 1A, the capillaries indicated by the thin lines are connected, forming a continuous tube. Separations such as isoelectric focusing (IEF) and isotachophoresis can be performed in this tube. After IEF, piece X slides to the other position as depicted in FIG. 1B to segment and isolate the pre-separated compounds. The segmented samples are then collected into different vials (without loss of any sample) for the 2^nd-D separation. Note: the pre-separated samples are collected on both piece X and piece Y.

FIG. 2A presents a schematic arrangement of the valve device assembly to enable piece X to switch back and forth as illustrated in FIG. 1. Pieces X and Y are held together by two blocks using bolts and nuts via four through holes (see FIG. 2C for the positions of the four holes). FIG. 2B exhibits a cross-section view and FIG. 2C displays the top-view of the block holding piece X. Referring to FIG. 2B, a pocket is machined on the bottom of the block. The pocket has a depth that is slightly (e.g. 1 mm) shallower than the thickness of piece X. This allows piece X to extrude out slightly to ensure good sealing between pieces X and Y when the assembly is tightened (see the interface between pieces X and Y in FIG. 2A). The length of the pocket equals to the length of piece X plus the sliding distance, which allows the ends of the pocket to serve as two stoppers for the valve switching. An open slit will be made in the middle of the Nylon block (see FIG. 2C) to permit the attached capillaries coming out. Two holes are drilled perpendicular to the open slit so that two pins can be inserted in to secure the two switching bars. The bottom Nylon block is used to hold piece Y in a fixed position. Referring back to FIG. 2A, by pushing the right switching bar to the right, piece X will move to the left and stop as it hits the left-end of the pocket. By pushing the left switching bar to the left, piece X will move to the right and stop as it hits the right-end of the pocket.

The following presents an example protocol to perform an IEF separation/fractionation.

• • 1. Prepare the IEF sample by mixing ampholytes with a protein sample
  • 2. Set the fractionation device to the position as depicted in FIG. 1A
  • 3. Load the sample into the continuous capillary (the thin-line of FIG. 1A)
  • 4. Run IEF
  • 5. Turn off the HV for IEF and switch the device to the other position as depicted in FIG. 1B
  • 6. Collect the proteins inside the segmented capillaries (Although we have “split” the sample into 100 fractions, proteins of similar pI should be “focused” in one or two fractions after IEF. That is, the resolution of IEF is retained.)

All of the methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the invention has been described with respect to the described embodiments in accordance therewith, it will be apparent to those skilled in the art that various modifications and improvements may be made without departing from the scope and spirit of the invention. For example, it will be apparent to those of skill in the art that variations may be applied to the methods and apparatus and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, it is to be understood that the invention is not to be limited by the specific illustrated embodiments, but only by the scope of the appended claims.

Claims

1. A device for high-resolution and high sensitivity assays, comprising:

at least a first block and a second block that are configured to slide relative to each other from a first position to a second position;

at least two tubes that are attached to at least one of the said blocks;

at least a semi-looped channel segment that that is attached to or embedded in at least one of the said blocks;

wherein at the first position, the device is configured so that at least two of the said semi-looped channel segments are joined together forming a single continuous channel, and

wherein at the second position, the device is configured so that the said continuous channel is broken into at least two channel segments, at least one of the segments is in fluidic connection with two tubes that are attached to one of the said blocks;