Patent Application
20070144907
This application claims priority to EP 05028551.9 filed Dec. 28, 2005.
The present invention refers generally to two-dimensional gel electrophoresis, and in particular to disposable electrophoresis device for the separation of a complex protein sample using two-dimensional gel electrophoresis and a method thereof.
Two-dimensional slab gel electrophoresis is still the most used approach to proteomics and it might be still for several years, despite alternative chromatographic methods are gaining popularity, if after improvement over the years, other limitations still present are addressed. In particular, this remains a time-consuming and laborious procedure, requiring trained personnel, on the hands of whom the quality of results is mainly depending. Just because there is much manual work involved, reproducibility is indeed difficult to achieve, whereas on the other hand gels are mostly made to be compared. Although running conditions can be quite reproducible, as these are controlled by proper set-up and power supplies, and new buffer systems have increased gel stability and performance, problems with accuracy and consistency can arise from variations in the other numerous parameters to keep under control. Some of these are for example, sample loading and rehydration, in terms of sample amount, losses, and homogeneity of the strip, strip handling with risk of damaging and contamination, imprecise and slow coupling of the strip to the gel, gel casting and polymerization, in terms of homogeneity, casting and reaction speed, especially for gradients, air sensitivity, time for completion until run is started, risk to trap bubbles causing consequently also field discontinuities, increase in temperature during the run, pH and viscosity changes, and loss of buffer capacity.
Describing in detail the entire gel electrophoresis process is out of the scope of this invention as several reviews can be retrieved in the literature. A brief overview is however given below to help in understanding.
Normally, first dimension separation consists of isoelectric focusing (IEF) where proteins separate according to their isoelectric point in a pH gradient, typically immobilized (IPG), in a long and narrow supported gel assuming the form and taking the name of a gel strip. The gel strips, commercially available, are normally supplied in a semi-dry state and they have to be rehydrated with the sample solution before analysis. This operation takes from a few hours to typically overnight and usually takes place under mineral oil to prevent drying and crystallization of urea present in the sample solution. IEF takes place also under mineral oil for the same reasons in the same or a different tray with the gel strip in contact with two electrodes at the sides, between which a high voltage is applied.
After IEF the gel strip has to be equilibrated, which means that the proteins focused within the gel strip have to be first alkylated and then complexed with sodium dodecyl sulfate (SDS) in order to be later transferred to a second dimension gel wherein the proteins are separated according to size. Reduction/Alkylation can be achieved by different reagents and one has the option to perform this step during sample preparation before rehydration such as suggested by Herbert et al. (Herbert B et al. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when and how? Electrophoresis 22, 2046-2057), although this might result in shifts of the isoelectric points. However SDS equilibration can be performed only after IEF, so that the gel strip is literally washed for several minutes in the equilibration solution containing SDS. The gel strip is then placed on top and in contact with a prepolymerized SDS polyacrylamide gel and coupling is achieved by pouring a hot agarose solution over the strip. This is usually accomplished between two glass plates which are clamped together and then placed in a buffer containing cassette where voltage is applied across the gel. The gel might be formed with a porosity gradient in order to increase resolution in the second dimension. After this is complete, the gel is removed, fixed, stained and background staining dye removed before proceeding eventually with the subsequent steps, i.e. spot picking, digesting, and mass spectrometry analysis.
Ideally, what is desirable is that no further manual intervention is required after the sample has been loaded, in a way similar to the instrumental chromatographic approach, the main strength of which is indeed automation associated with better reproducibility. Automation and integration of the steps involved in the gel-based procedure is a challenge that others in the art are also addressing.
An integrated, fully automated system, mimicking step by step the manual procedure, including also sample preparation and gel strip casting is described in U.S. Pat. No. 6,554,991 B 1. The robotic machinery behind it, the complexity of the operation and the investment necessary go however far beyond a practical and widespread use of it, especially among the smaller research laboratories.
Published US patent application No. 2003/0127331 discloses a system where the gel strip once cast at the bottom of a vertical mold formed by two plates doesn't have to be moved after IEF. It is understood that the gel strip can be treated with the equilibration solution, apparently just from one side, and subsequently coupled to the second dimension gel by pouring the gel solution into the mold directly in contact with the gel strip or on top of an agarose layer. Doubts remain however concerning the efficiency and/or the time of the equilibration with the SDS having to diffuse inside the gel strip just from one side and whether the resolution obtained in the first dimension can be preserved. As no mention is made concerning the polymerization method, the long times associated with the classical method increase further the concern about loss of resolution. Also, the way the gel strip is formed and the sample is added is less reproducible and the fact that a sealing tab at the bottom of the mold has to be removed at the end is not practical.
In EP 0366897 it is disclosed that the gel strip is first separated from a prepolymerized gel by means of a non-conductive phase-change material, which is melted after IEF by increasing the temperature, removed and substituted with other gel medium. No reference is however made to the equilibration step and besides the concerns about the effect of the temperature for proteins and gel, remains the problem associated with closing and opening this time the top of the mold.
Other barrier means between a gel strip and gel are disclosed in WO 02/084273 A1. The first embodiment reported therein making use of sliding solid barriers is certainly not the most advantageous as formed gels might be disrupted by this action. A more interesting solution makes use of pneumatically assisted valves consisting of soft and expandable material, separating the strip from a preferably precast gel. The space occupied by the valve is later filled with agarose for coupling. In a third embodiment, semi-walls at the sides of the gel strip, are used as gasket against which a foil used also as gel support can be pressed, thus opening and closing the strip chamber by changing position relative to the opposite rigid surface. The gel solution is in this case introduced and polymerized preferably after opening the strip chamber at the end of the first dimension or, with difficulty to imagine as the foil has to move, the gel can be precast. Although the possibility to immerse the gel strip with the gel solution in one step is disclosed, the use of agarose is again preferred. Equilibration solutions can access the gel strip through the rigid part of the device.
There are, however, weak points still left in the above disclosed system, first of which is represented by dead volumes for the sample regardless of the embodiment. Indeed, an extra space is necessary above the fully rehydrated gel strip in order to allow the flow of the equilibration solution after the first dimension, thus requiring excess of sample to fill that same gap when rehydrating the gel strip. Also, the fact that excess liquid is left above the gel strip during IEF can result in disturbed focusing and horizontal striking, and if removed can result in drying of the gel strip. Moreover, loss of resolution due to the long waiting time for gel polymerization and deleterious effects due to penetration of acrylamide monomers into the gel strip can be expected when casting the gel after IEF, as no methods of polymerizing the gel solution, other than the intended classical one, are claimed or even mentioned. This must be the reason why the use of agarose is preferred in all cases. Agarose brings however new annoying issues, like the need to be boiled before melting and the troubles to remove it from all the tubing and fittings once it has started to gel. Concerns remain also regarding the efficacy in maintaining an even and non deformed foil shape, important in guaranteeing a homogeneous gel thickness.
It is against the above background that the present invention provides an improved disposable device and ways of processing the same. The present invention provides convenient and effective means of integrating the generally accepted and manually executed steps for the separation of a complex protein sample in proteomics analysis based on two-dimensional gel electrophoresis. In particular, the present invention provides a system having a disposable electrophoresis device for sample separation based on two-dimensional electrophoresis, which requires only minimal or preferably no manual intervention once the sample has been loaded to the device.
In one embodiment, a disposable electrophoresis device for separation of a complex protein sample using two-dimensional gel electrophoresis is disclosed. The device comprises a carrier with a first area for a first dimension gel strip and a second area for a second dimension gel where the two areas are directly in contact with each other; and a body made of a hard component and a flexible component layer, the body being provided at a variable distance from the carrier, the body defining at least one cavity used for external actuation of at least one valve, wherein closing of the valve is represented by a reversible stretching of the flexible component layer in a direction towards the carrier upon external actuation of the valve through the cavity.
These and other features and advantages of the present invention will be more fully understood from the following description of various embodiments of the present invention taken together with the accompanying drawings.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
a and 1b show schematically and not to scale, embodiments according to the present invention having a two-component disposable core part with closed valves and two variable thicknesses, one during rehydration and IEF (
a and 2b show schematically and not to scale, two embodiments according to the present invention for a two component disposable core part during gel-casting in the second dimension.
a and 3b are flow diagrams of a process for sample separation based on two-dimensional electrophoresis according to the present invention.
Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
Below and with reference to the following schematic drawings a brief description of examples of systems and processes according to the present invention are disclosed.
A two-dimensional electrophoresis system, generally indicated by reference symbol 100 in
A body 2 of the electrophoresis device 1, which includes at least one valve (e.g., valves 9 and 10), is injection molded by e.g. applying component molding technology, meaning that no assembly is needed between the parts and thus reducing the cost of manufacturing. In one embodiment, a generally rigid, inflexible or hard component 3 of the body 2 is a material chosen among acrylic glass (i.e. polymethylmethacrylate (PMMA)), polycarbonate (PC), polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET or PETE), or cyclo-olefin copolymers (COC) or any other suitable polymer material which can be molded and that is UV transparent, with the specific intent to allow UV-initiated fast polymerization of the gel solution during the process by shining light of appropriate wavelength directly through it, from one or multiple lamps integrated into the instrument 30. Another component of the body 2 is a first soft, flexible, or elastic component layer 11. In one embodiment, the first elastic component layer 11, which portions thereof defines valves 9 and 10 of the device 1, is a material chosen among thermoplastic elastomers (TPE) that is compatible with the previously chosen body material 3 such as, for example, PTS—Thermoflex® (Plastic Technology Service Ltd, Salisburg SP5 4BZ, UK), Santoprene® (Advanced Elastomer Systems, L.P., Akron, Ohio, USA), or any other suitable elastic material that can be molded in a convenient flat laminar shape and be directly attached to an inner surface of the hard component 3 of the disposable body 2 and to which a second dimension gel (discussed hereafter in a later section) does not stick. In one embodiment, in addition to the above requirements, the first elastic component layer 11 may further be a UV transparent material. By defining two slits or cavities 40a and 40b through the disposable body 2 immediately flanking a first zone provided for a gel strip laying underneath, the first elastic component layer 11 can be stretched down by external rigid actuation performed by the instrument 30, such as via inserts 12 and 14 of the instrument 30, thus trapping a gel strip 7 in a closed and tight environment (i.e., a strip chamber 32) by pressing against a gel strip carrier 5. The carrier 5 is a material selected from a polymer film, a foil, or a glass plate. In one embodiment, the carrier 5 is provided with a gel-bond material such that the second dimension gel can be firmly attached thereon. Similarly, in other embodiments, slits or cavities can be designed also in other positions along the hard component 3 of the body 2 to divide a gel chamber 16 defined between the carrier 5 and the first elastic component layer 11 of the device 1 into compartments if needed or simply to close any eventual open edge.
A second elastic component 17 is also integrated in the disposable electrophoresis device 1 as a gasket or ring provided along the perimeter of the area defining the gel chamber 16 (i.e., a second area for the second dimension gel). The second elastic component 17 is provided in sandwich arrangement between the inner surface of the body 2 and the carrier 5. In one embodiment, the second elastic component 17 is molded at the same time and with the same procedure, and also with the same material as the first elastic component layer 11. The second elastic component 17 is provided for the function of allowing variation (i.e., increasing and decreasing) of the distance between the two opposite surfaces of the first elastic component layer 11 and the carrier 5 upon external applied pressure 38, such as provided by the processing instrument 30. In one embodiment, the distance between the two opposite surfaces of the first elastic component layer 11 and the carrier 5 corresponds to about 0.7 mm during rehydration and IEF, which is indicated by symbol a in
In one embodiment, the carrier 5, which cross-links to the gel during polymerization, is laminated to the disposable device 1 and can be peeled off by the user after the process.
In addition, in one embodiment, the outer dimensions of the disposable device 1 are designed according to an industry standard in order to facilitate robotic handling, and in one particular embodiment is according to the ANSI SBS (American National Standards Institute, The Society of Biomolecular Screening) standard with dimensions of: 127.76±0.25 mm×85.48±0.25 mm.
In one embodiment, disposable electrodes (not shown) consisting of an inexpensive material, such as for example, graphite, conductive paper, etc., can be integrated in the disposable device 1 for the first dimension separation. In the illustrated embodiment of
In another embodiment, in order to make the disposable device 1 even simpler and more compact, buffer reservoirs 44a and 44b for the second dimension are part of the instrument 30 and clamped to the disposable when needed by simple means making use of a gasket (not shown) and external pressure. In order to prevent the loss of buffer capacity associated with small reservoirs and small buffer volumes, in one embodiment the buffer is freshly circulated from larger reservoirs (not shown) upstream. In order to prevent keeping the entire circuit under the applied voltage during buffer replacement, in one embodiment, a “stop and go” discontinuous approach by opening the circuit defined between the electrodes 42a and 42b at intervals to replace the buffer is used In another embodiment, buffer replacement is achieved by restricting the channel of communication between large and small reservoirs, eventually also dispensing air bubbles along the liquid path as insulators. In an alternative embodiment, the limitation of the buffer capacity is overcome by recirculating the cathode buffer with the anode buffer and vice versa. By this means, the buffer reservoirs 44a and 44b can be kept small with no additional need of buffer during the run.
In order to maintain the flatness and evenness of the carrier 5, important especially to guarantee gel homogeneity and efficient cooling, both fundamental for reproducibility, a cooling block 34 of the instrument 30, on which the disposable device 1 geometrically fits, such as for example, via the external side of the carrier 5 contacting thereon, is provided. The cooling block 34 in one embodiment is made of porous ceramics, and in other embodiments is another porous or holes containing material such as metal, other heat-conductive alloy, polymers, and combinations thereof through which a vacuum suction, indicated by reference symbol 46, can be applied. At the same time this represents an advantageous way to steadily fix the disposable device 2 into the instrument 30 so that in one embodiment it is rotated about 90° during gel casting by mechanical rotation, indicated by reference symbol 48, of the cooling block 34.
In one embodiment, the disposable device 1 is generic containing no IEF strip, i.e., gel strip 7, thus leaving the freedom to the operator to insert a desired strip with a desired pH range, and avoiding the need to deliver and store the entire disposable device 1 with the strip inside at refrigeration temperature. Guiding features are provided so that no misplacing can occur, e.g. with closed valves, while the electrodes 36a and 36b in one embodiment serve also to keep the gel strip 7 in place. In another embodiment, with the disposable device 1 made simpler and compact as described above, the gel strip 7 is integrated into the disposable device 1, so that one could order different sets of disposables containing different strips. In still another embodiment, the gel strip 7 is already attached on the carrier 5, with the carrier 5 being delivered separately from the disposable body 2. In such an embodiment, the user then has to assemble the two parts together, e.g. with the help of positioning holes. In still yet another embodiment, the gel strip 7 is inserted through an opening in the carrier 5, which is provided as a bottom cover to the body 2, which is then closed afterwards with a tape-like mechanism.
Another element applicable to the embodiments of the device 1 with valves, is the use of membranes or a blade at a gel/buffer interface 50. What is disclosed in the illustrative embodiment shown by
Process steps, which in one embodiment are automatically executed by the processing instrument 30, are described hereafter with reference to the attached figures.
First, in step 310 (
Second, in step 312, a protein sample (not shown) for separation is provided with a rehydration solution (not shown) to the disposable 1 with the two valves 9 and 10 at both sides of the gel strip 7 closed. It is to be appreciated that a strip chamber 32, which is defined between the closed valves 9 and 10, and which has one dimension being distance a, is filled with the protein sample/rehydration solution during this filling step.
Third, in step 314, a waiting period is executed for rehydration of the gel strip 7 within the strip chamber 32. In one embodiment, the rehydration time is at least one hour, and in other embodiments may be any time needed to rehydrate the material used for the gel strip 7. In addition, during rehydration, the temperature of a cooling block 34 of the processing instrument 30 provided adjacent the disposable device 1 is set at about 30° to about 35° Celsius.
Fourth, in step 316, IEF is run by applying ramping high voltage between the two opposed electrodes 36a and 36b either integrated or inserted at this moment by the processing instrument 30. During IEF, the instrument 30 controls also the temperature of the cooling block 34 which in one embodiment is set at about 20° Celsius.
Fifth, in step 318, distance a as shown in
Sixth, in step 320, the alkylation/SDS equilibration solutions are flown into the free space or channel 23 created on top of the strip 7 and empty at last. The free space or channel in one embodiment is about 0.3 mm.
Seventh, in step 322, the whole disposable 1 is rotated by about 90° to bring it in a more or less vertical position as shown in
a shows the design of the disposable as shown in
Eighth, in step 324 (
Ninth, in step 326, the gel solution for forming the gel of the second dimension separation is provided through e.g. a hole (not shown) in the disposable body 3 for casting, thus achieving at the same time coupling with the strip 7. The gel solution is polymerized by a fast UV initiated reaction which is completed e.g. in less than 5 minutes. Polymerization is possible with the present invention due to the disposable body 3 (fully or partially) and layer 11 being transparent to UV radiation. If desired, gradient gels can be also cast in a similar manner.
Tenth, in step 328, valve 10 is opened within the arrangement of
Eleventh, in step 330, eventually the arrangement is rotated back to a horizontal position as in
Twelfth, in step 332, the running buffer is introduced and the second dimension run is initiated by applying voltage to the electrodes (not shown) between the two reservoirs. The electrodes are either integrated into the disposable or in the instrument. In one embodiment, the second dimension run is conducted at a controlled temperature such as, for example, 20° Celsius. The running buffer is circulated as necessary.
Thirteenth, in step 334, after the second dimension run is completed, the disposable 1 is removed from the instrument and opened to remove the gel by pealing off the foil 5, to which the gel is bonded.
The above described process with reference to the attached drawings is of course an example suitable for describing the present invention and is not at all limiting to the present invention. The type of material used for producing the disposable device 1, including the elastic component layer 11 for the valves, the carrier 5, compressible or elastic parts 17, etc., could be changed in an appropriate manner. The use of a UV or light transparent material for the disposable device 1 is preferred so that UV or light initiated polymerisation of the second gel in the gel chamber 16 of the device 1 is possible, but it should not be a limiting factor to the present invention. Furthermore using two, three or more valves is possible. One feature of the present invention of course is, that the distance between the inner opposing surfaces of layer 11 and the carrier 5, on which the first gel strip 7 is attached is variable, which means that after the first dimension separation, the distance therebetween can be expanded, for example, due to the arranged compressible component or gasket 17.
The combination of at least one valve 9 or 10 around the gel strip 7 with the variable distance between the inner opposed surfaces of the carrier 5 and the elastic component layer 11 (e.g., about 0.7 mm and about 1.0 mm during first and second dimension respectively) raises the number of allowed positions for the at least one valve 9 or 10 from two to three and offers advantages compared to the prior art. First of all, no or minimum sample excess is required to rehydrate the gel strip 7 as the volume of the strip chamber 32 created by the valves 9 and 10 corresponds to the volume of the rehydrated strip. In this way, also IEF can be run under optimal conditions with no liquid excess on top of the gel strip 7. Space is created on top of the gel strip 7 only after IEF to introduce flowing equilibration solutions, thus providing also optimal equilibration conditions. Finally, as a thicker gel with a small space above the gel strip 7 is required to achieve proper coupling and perform a good second dimension analysis in terms of field homogeneity, 2D resolution and reproducibility, optimal conditions are provided also in this subsequent step.
Unlike the prior art, UV-initiated fast polymerization is adopted in the field of two-dimensional gel electrophoresis, choosing an initiator that is stable in the acrylamide gel solution until exposed to a light source whose wavelength range comprises its absorbance spectrum. Valves 9 and 10 are used only to close the gel strip 7 in a tight or closed environment, and not as barriers between the gel in the gel chamber 16 during the second dimension separation and the gel strip 7 in the strip chamber 32 because the gel can be polymerized quickly after the first dimension and doesn't have to be precast. Thus chemistry, storage time and conditions as well as waiting time for post- or pre-IEF polymerization are no longer an issue. Because polymerization proceeds fast in the present invention, such as from using the method disclosed in commonly assigned U.S. patent application Ser. No. 11/278,975, the disclosure of which is herein incorporated fully by reference, the use of a coupling gel such as, for example, agarose can also be eliminated. The gel solution can now fill completely the gel chamber 16, contacting, covering and enclosing the gel strip 7 therein, while this is not possible with the traditional method, making use of ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) as initiator and catalyst respectively of radical polymerization. These reagents indeed have to be added and mixed at the last moment as they start immediately polymerization already during casting, thus causing already preparation problems, and because the reaction proceeds slowly taking normally more than one hour to be completed, loss of resolution obtained during the first dimension and diffusion of acrylamide monomers into the strip, causing possible cross-linking with the proteins, become other important issues. For the same reasons, fast UV polymerization becomes also particularly convenient when casting gradient gels.
Although the various embodiments of the present invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP 05028551.9 | Dec 2005 | EP | regional |
