Patent Application
20070205106
The present invention relates to an electrophoresis apparatus and methods of its use for fractionation of a complex sample.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
Electrophoresis has been widely applied in separating proteins, nucleic acids, and other charged molecule species for analytical or preparative purposes, and also in the analytical or preparative fractionation of heretogeneous populations of dispersed cells or other types of macroscopic particles. In the analysis of complex ampholytic samples, such as in proteomics, it would often be desirable to reduce the complexity of a sample by pre-fractionation. Two dimensional electrophoresis (2DE) is believed to be currently the most commonly used separation method in proteomics. In the first dimension of 2DE, conventional gel isoelectric focusing (CGIEF) or better yet, immobilized pH gradient IEF (IPGIEF) are used to separate proteins according to their pI values.
Both CGIEF and IPGIEF have numerous practical problems including a limited sample loading capacity, a limited dynamic range, precipitation of proteins during IEF separation (streaking) and an inability to tolerate a large amount of salts in the samples. P. G. Righetti et al., (Electrophoresis 21, 2000, 3639-3648); P. G. Righetti et al., (Anal. Chem. 73, 2001, 320A-326A) and D. W. Speicher et al., (Anal. Biochem., 284, 2000, 266-278); X. Zou & D. W. Speicher, (Proteomics, 2, 2002, 58-68) have shown that pre-fractionation of a complex protein sample in a multi-compartmental electrolyzer significantly improves the performance of 2DE. It is believed that the common limitation of both the ISOELECTRIQ2™ unit, marketed by Proteome Systems™ and the ZOOM™ unit, marketed by INVITROGEN™ is two-fold. First, the distance between the center of the separation compartment and its walls is relatively large (greater than about 5 mm), and second, the electrophoretic migration distance in each compartment is long, about 25 mm and 13 mm, respectively. The first, coupled with the fact that the separation compartments are made of thermally insulating polymers, leads to poor Joule heat dissipation and severely limits the electric power that can be applied to the system (max. 5 W and 3.5 W, respectively). The second, coupled with the low electrophoretic mobilities brought about by the low field strength, a consequence of the limited heat dissipation capability of the systems and the long electrophoretic migration distance, leads to slow separation velocities. Consequently, the fractionation times in these systems are long, 6 to 16 hours and 4 hours, respectively. Both systems use compartments with relatively large volumes (about 5 ml and 0.7 ml for each compartment, respectively), and the volume of the compartments cannot be easily reduced.
Gradipore Limited (Life Therapeutics) developed a small scale electrophoresis unit for size-based and charge-sign-based fractionation of complex samples (WO 01/8878, incorporated herein by reference). It is believed that, in practice, active cooling of at least the electrolytes was required to prevent over-heating of labile proteins during electrophoresis. Gradipore subsequently developed a scaled-down version of the GRADIFLOW™ electrophoresis unit, for size-based and charge-sign-based pre-fractionation of complex samples. In this MICROFLOW™ system, about 3 cm×4 cm polymer frames, separated by polyacrylamide membranes, are stacked next to each other to form the separation compartments and contain stagnant sample solutions. The compartment stack is terminated at both ends by a large volume anode compartment and cathode compartment. It is believed that in practice, the anolyte and catholyte are cooled and circulated through these compartments to provide convective heat removal.
Slow separation speed of the currently known electrophoresis systems, specifically, isoelectric fractionation systems, useful as they are, are believed to be due to the failure of existing systems to sufficiently address three interrelated design limitations. The first speed limitation comes from the fact that as the ampholytic components of a sample approach their isoelectric state, their electrophoretic mobilities approach zero. Consequently, when the components are close to their isoelectric state, they need an increasingly longer time to move across a certain distance. The second speed constraint comes from mechanical design problems that limit how short the electrophoretic migration path and how small the volume of the individual compartments holding the sample solutions can be before mechanical assembly and leak-tight sealing of the compartments become very difficult. The third performance limitation comes from the amount of Joule heat that is produced during electrophoresis. Since Joule heat dissipation occurs through the walls of the separation compartment, and since heat must first be transported from the separation medium to the wall, both of which are inefficient processes, the amount of Joule heat produced during fractionation must be limited and external, active cooling means must be applied. This means that the electric power input into the system to effect a separation must be limited. This results in a low electric field strength which, in turn, results in slow electrophoretic migration velocities and concomitant long separation times presently observed with current apparatus.
Accordingly, there exists a need for an electrophoresis apparatus or device suitable for processing small volume samples while effectively dissipating the heat generated during electrophoresis and reducing separation times. More specifically, the second and third speed limitations discussed above can be eliminated or negated to a great extent by selecting a structural material for the separation compartments of an electrophoresis apparatus that is a good electrical insulator yet has a relatively high thermal conductivity and specific heat. From these materials, one could make separation compartments that act as high capacity heat sinks by creating small separation compartments with appropriately selected characteristic dimensions in relatively large pieces. These heat sinks would greatly mitigate the need for active external cooling and/or for the reduction of the electrophoretic power used.
The present invention relates to an electrophoresis apparatus and methods of its use for fractionation of a complex sample. The apparatus more specifically relates to Membrane-Separated Wells for Isoelectric Focusing and Trapping (MSWIFT). Primary application areas of MSWIFT and its modes of operation are in the analytical-scale fractionation of complex samples, such as pre-fractionation of protein samples for proteomic analysis, preparation of fractions for mass spectral (MS) analysis, bioactivity testing, enzymatic analysis, etc., rapid selection of isoelectric membranes for preparative-scale isoelectric trapping (IET) separations, and characterization of isoelectric membranes.
The present invention provides for an electrophoresis apparatus for characterizing, measuring and/or altering a composition of a sample. The apparatus comprises an anode and a cathode, the cathode spaced from the anode so as to define a distance along a longitudinal axis, the anode and cathode further defining an electric field having a direction substantially along the longitudinal axis. The apparatus includes an anode compartment, the anode disposed therein and a cathode compartment, the cathode disposed therein. Each of the anode compartment and the cathode compartment can be configured to hold at least one electrolyte, and at least one of the anode compartment and the cathode compartment can be configured to hold at least a portion of the sample. Each of the anode compartment and the cathode compartment includes means for addition or removal of a solution, a first compartment dimension, a second compartment dimension, and a third compartment dimension. The first compartment dimension can be substantially orthogonal to the direction of the electric field, and the second compartment dimension can be substantially orthogonal to the direction of the electric field and the first compartment dimension. A ratio of the first compartment dimension and the second compartment dimension defines an aspect ratio of the compartment, and the third compartment dimension can be substantially parallel to the direction of the electric field and substantially orthogonal to the first and second compartment dimensions. The apparatus further comprises an ion-permeable barrier positioned between the anode compartment and the cathode compartment. The ion-permeable barrier can be configured to prevent convective mixing between compartments. At least a portion of at least one of the anode and cathode compartments can be made of an electrically insulating material having a thermal conductivity greater than about 1 W/mK and a specific heat greater than about 100 J/kgK and the aspect ratio of at least one of the anode compartment and the cathode compartment can be less than one.
The electrophoresis apparatus preferably further comprises sealing means disposed between the anode compartment and the cathode compartment. The sealing means is preferably adapted to contain the ion-permeable barrier and provide access of ions to the ion-permeable barrier. Wherein the sealing means is made of a water insoluble polymer, the polymer can be natural or synthetic. Preferably, the water insoluble polymer of is selected from the group consisting of polyethylene, polypropylene, polyisobutylene, polyalkylenes, polyfluorocarbons, poly(dimethylsiloxane), poly(dialkylsiloxane), poly(alkylarylsiloxane), poly(diarylsiloxane), poly(ether ketones) or a combination thereof.
The electrophoresis apparatus further preferably comprises housing means for containing the anode and cathode compartments. Preferably, at least a portion of the housing means is made of a material having a thermal conductivity greater than about 1 W/mK and a specific heat greater than about 100 J/kgK. Moreover preferably, material of the at least portion of the housing means can be selected from the group consisting of alumina, aluminum nitride, zirconia, zirconium nitride, boron nitride, silicon nitride, silicon carbide, ceramics, fused silica, quartz, glass or any combination thereof.
The electrically insulating material of the at least one part of the anode or cathode compartment can be preferably selected from the group consisting of alumina, aluminum nitride, zirconia, zirconium nitride, boron nitride, silicon nitride, silicon carbide, ceramics, fused silica, quartz, glass or any combination thereof.
Preferably, the ion-permeable barrier is essentially free of weekly acidic functional groups or weakly basic functional groups or anionic functional groups or cationic functional groups. Alternatively, the ion-permeable barrier can be an isoelectric barrier.
In an alternative embodiment of an electrophoresis apparatus according to the present invention for measuring, characterizing, or altering a composition of a sample, the apparatus comprises an anode and a cathode, the cathode spaced from the anode so as to define a distance along a longitudinal axis, the anode and cathode further defining an electric field having a direction substantially along the longitudinal axis. The apparatus includes an anode compartment having an anode disposed therein and a cathode compartment having a cathode disposed therein. At least one separation compartment is preferably positioned between the anode and cathode compartments. Each of the anode compartment, cathode compartment and at least one separation compartment can be configured to hold at least one electrolyte. At least one of the anode compartment, cathode compartment and at least one separation compartment can be configured to hold at least a portion of the sample, and each of the anode compartment, cathode compartment and at least one separation compartment includes means for an addition or removal of a solution, a first compartment dimension, a second compartment dimension, and a third compartment dimension. The first compartment dimension can be substantially orthogonal to the direction of the electric field, the second compartment dimension can be substantially orthogonal to the direction of the electric field and the first compartment dimension. A ratio of the first compartment dimension and the second compartment dimension defines an aspect ratio of the compartment, and the third compartment dimension is preferably substantially parallel to the direction of the electric field and substantially orthogonal to the first and second compartment dimensions. The apparatus further includes an ion-permeable barrier positioned between each of the anode compartment, the at least one separation compartment and the cathode compartment. The ion-permeable barrier can be configured to prevent convective mixing therebetween. At least a portion of at least one of the anode compartment, the cathode compartment and the at least one separation compartment is made of an electrically insulating material having a thermal conductivity greater than about 1 W/mK and a specific heat greater than about 100 J/kgK and the aspect ratio of at least one of the anode compartment, the cathode compartment and the at least one separation compartment is less than one.
The present invention further provides for a method of altering a composition of a sample by electrophoresis which includes providing an electrophoretic apparatus according to the present invention. The method further includes selecting an ion-permeable barrier for use between the anode and cathode compartments, providing an electrolyte to the anode compartment, providing an electrolyte to the cathode compartment, providing at least a portion of a sample to at least one of the compartments, creating an electrophoretic direct current between the anode and the cathode by applying an electric potential between the anode and the cathode, and causing a transfer of at least one part of at least one component of the sample across the ion-permeable barrier. Alternatively, a method according to the present invention can include providing at least a portion of a sample to at least one of the compartments of an apparatus according to the present invention, providing at least one electrolyte to any of the compartments free of a sample component, creating an electrophoretic direct current between the anode and the cathode by applying an electric potential between the anode and the cathode, and causing a transfer of at least one part of at least one component across an ion-permeable barrier.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate an embodiment of the invention, and, together with the general description given above and the detailed description given below, serve to explain features of the invention.
Shown in
In the embodiment shown in
Preferably disposed between anode and cathode compartments 14, 15 and within housing means 1 can be one or more separation elements defining or forming separation wells or compartments 40. Although specifically shown in
Housing means 1 orients and seals anode compartment 14, cathode compartment 15 and where provided, separation compartment 40 such that compartments 14, 15 and 40 are substantially aligned along longitudinal axis A-A so as to facilitate communication therebetween in which components of the solution to be altered can migrate between compartments 14, 15 and 40 under the influence of the electric field. In order to prevent fluid loss from compartments 14, 15 and 40 to the environment, apparatus 10 can further include sealing means 12. Preferably, housing means 1 is configured so as to permit top loading of anode, cathode, and separation compartments 14, 15, 40 and sealing means 12 into housing means 1.
Referring again to
In order to facilitate the sealing action of sealing means 12, housing means 1 can include axially opposed compression members 8, 9, preferably formed from an electrically insulating, non-brittle, sufficiently rigid material, such as PVC material, that can be axially displaced along longitudinal axis A-A to compress anode and cathode compartments 14, 15, sealing means 12, ion-permeable barriers 18, and where present, separation compartments 40. In addition, axial displacement of opposed compression members 8, 9 facilitates removal and/or replacement of the individual anode, cathode and separation compartments 14, 15, 40, sealing means 12 and ion-permeable barriers 18 from housing means 1. Compression members 8, 9 can directly act on axially opposed end plates 16, 11 which are each preferably engaged with sealing means 12 to transmit the compressive force to the assembled anode and cathode compartments 14, 15, separation compartments 40, sealing means 12 and ion-permeable barriers 18. Compression members 8, 9 can include a threaded rod and nut assembly 5 so as to axially displace compression members 8, 9 along longitudinal axis A-A, however it is to be understood that other means of linear displacement may be provided.
Preferably, at least a portion of housing means 1 is made from a material having a thermal conductivity greater than about 1 W/mK, and a specific heat of greater than about 100 J/kgK, preferably greater than about 250 J/kgK, and especially greater than about 500 J/kgK. Referring to
Specifically shown in
As seen in
It is to be understood that cathode compartment 15 and any number of separation compartments 40 of apparatus 10, 10′ or 10″ can be independently similarly or variably configured in a manner as described herein with respect to anode compartment 14. More specifically, the aspect ratio of anode compartment 14 can be different from the aspect ratio of cathode compartment 15 and/or separation compartments 40 by varying first and second dimensions a, b of the respective compartments provided the aspect ratio of the respective compartments remains less than one. In a preferred embodiment, separation compartment 40 can be formed by grinding a 1.5 mm wide, 5 to 45 mm deep groove into 99.8% nonporous alumina blocks. Alternatively, grooves can be formed in alumina blocks as thin as 0.25 mm and as thick as 2.5 mm.
Preferably first dimension a and third dimension c are minimized. Minimizing first dimension a can in turn minimize the distance in the solution through which heat can be conducted to the wall of anode compartment 14, cathode compartment 15 and/or separation compartment 40. Minimizing third dimension c can mean that for a given applied potential, the electric field strength, and consequently the electrophoretic migration velocities of the components of the sample being processed are high, thus reducing the required separation time. Moreover, by minimizing third dimension c, i.e., the migration distance in a particular compartment, the overall distance from anode compartment 14 to cathode compartment 15 is minimized and therefore the separation time in processing the sample is once again further reduced. For example, all else being equal, if one replaced a 9 mm I.D., 10 mm long cylindrical separation compartment (with a volume of approximately 636 μl) by a 2 mm by 2 mm by 154 mm rectangular well with a volume of approximately 616 μl (migration distance×width×height of the well), the separation time would decrease about 25-fold (five times due to the reduced migration distance and five times due to the five-fold higher electric field strength for a constant applied potential). Additional benefits would accrue from the smaller temperature rise in the separation well brought about by the smaller heat conduction distance (4.5 mm vs. 1 mm). Preferably, so as to facilitate minimization of first and third dimensions a, c, apparatus 10, 10′, 10″ is preferably configured such that compartments, 14, 15, 40, sealing means 12 and ion-permeable barriers 18 are substantially axially aligned within housing means 1.
Again referring to
Third dimension c defines the migration distance of a component through anode compartment 14, cathode compartment 15 or separation compartment 40. Referring to
Second dimension b or depth of the compartment permits the use of open (from the top) compartments 14, 15 or 40, and provides for variable (partial) filling of compartments 14, 15 or 40 between zero and their respective full volume. There is no theoretical limit to the magnitude of second dimension b of the separation compartment orthogonal to the directions of both the electric field and first dimension of the compartment. Second dimension b can be varied to increase or decrease the required maximum reception volume of compartment 14, 15 or 40, without degrading the separation speed or the thermal characteristics of apparatus 10, 10′, 10″. Second dimension b can be varied by varying the dimensions of the material used to form compartment 14, 15 or 40, in conjunction with control of the grinding operation forming compartment 14, 15 or 40. Second dimension b can be as shallow as 5 mm and as deep as 40 mm. Shown in
First dimension a, second dimension b and third dimension c of each anode compartment 14, cathode compartment 15, and separation compartment 40 defines a reception volume for each to hold a volume of solution containing a sample component and/or an electrolyte. Preferably, anode and cathode compartment 14, 15 and where applicable, separation compartment 40, of apparatus 10, 10′, 10″ can receive a small volume of a solution containing an electrolyte and/or a sample component, the volume being less than about 5 ml, preferably less than about 2 ml, and more preferably between about 0.5 ml to about 0.001 ml.
In one alternative embodiment (not shown) of apparatus 10 shown in
Referring again to
Other geometries of sealing means 12 are possible. A preferred alternative embodiment of sealing means 12 is shown in
Sealing means 12′ includes an opening 13 for providing an access through which ions in a solution contained in anode compartment 14, cathode compartment 15 and where present, separation compartment 40 can migrate to and access ion-permeable barrier 18. Opening 13 is preferably substantially rectangular and includes a first characteristic dimension a′ substantially corresponding to first dimension a of anode compartment 14, cathode compartment 15 and where present, separation compartment 40.
Ion-permeable barrier 18 facilitates alteration by electrophoresis of a composition of a sample contained in one or more of anode compartment 14, cathode compartment 15 and separation compartment 40 of apparatus 10, 10′, 10″ of
For size-based separations, ion-permeable barrier 18 is preferably made from polyacrylamide and preferably has a nominal molecular mass cut-off from about 1 kDa to 1500 kDa. The molecular mass cut-off of the membrane material selected for ion-permeable barrier 18 will depend on the sample being processed and the type of components in the sample.
For IET-based separations, at least one ion-permeable barrier 18 can be an isoelectric membrane formed from any suitable material. Examples include, but are not limited to, copolymers formed from acrylamide, bisacrylamide, acrylamido weak electrolytes and acrylamido strong electrolytes. Preferably, the membranes are thin or ultra-thin, having a thickness of about 2 mm or less, preferably about 1 mm or less, and especially about 0.2 mm or less. Where ion-permeable barrier 18 is an isoelectric membrane, barrier 18 is provided with a concentration of buffering species in the membrane material. The isoelectric membrane forming ion-permeable barrier 18 does not have to be thick to provide adequate buffering capacity. As long as the isoelectric membrane forming ion-permeable barrier 18 can mitigate convective mixing between the contents of adjacent compartments 14, 15 and 40, the thinner the membrane, the shorter the distance the ampholytic components must travel. Therefore, thin isoelectric membranes can lead to shorter separation times. Also, for all else being equal, the thinner the isoelectric membrane, the less potential drops across it, and thus the less power is consumed to effect the electrophoretic separation. Additionally, most solutions used for rehydration of the IPGIEF strips contain 0.1-1% carrier ampholytes. In EEF pre-fractionation of proteomics samples, the fractions typically do not contain a single isoelectric species with a single pI value, rather many components that cover a relatively wide pI range (0.1<pI<2). This means that in the fractions, even at the end of the separation, the carrier ampholyte and ampholytic sample molecules are typically not in their isoelectric state, but are protonated and deprotonated by each other. This also means that in these fractions the ionic strength is higher than at the end of an IET separation in which pure, single components are produced in a compartment. If, due to the improved heat dissipation performance of electrophoresis apparatus 10 one could add, in a sufficiently high concentration, carrier ampholytes or auxiliary isoelectric buffers to the sample prior to electrophoresis, one could significantly increase the ionic strength in the respective fractions. This would improve protein solubility and increase the total amount of material that can be loaded or processed in the given volume of the system.
The characteristics of ion-permeable barrier 18 used depend on the sample and the type of separation or treatment contemplated. Within a single apparatus 10, 10′ or 10″, ion-permeable barriers 18 used may each be variably configured in a manner described herein to suit the electrophoresis application as needed. Ion-permeable barriers 18 or membranes can be purchased for use in the apparatus or made by the user prior to carrying out the desired electrophoresis run.
Referring again to
In one exemplary assembled embodiment of an electrophoresis apparatus 10, unfilled polycarbonate, for example, LEXAN® available from BOEDEKER PLASTICS, TX, is used to form housing means 1 and a ½ inch diameter borosilicate glass rod is used to form five separation compartments 40, each having a holding volume of 50 μl. Ion-permeable barriers 18 are formed from isoelectric membranes which are installed between sealing means 12 formed from silicone disks which reduce solution loss from membrane wicking. Such an assembled apparatus 10 could be used for the separation of low molecular weight pI markers and proteins and for UV-active carrier ampholyte-based membrane characterization. Using a surface treatment with a hydrophilic polymer on sealing means 12 can further reduce leaking problems and mitigate electroosmotic flow.
In another exemplary embodiment of an electrophoresis apparatus 10, housing means 1 is preferably constructed from LEXAN® and seven separation compartments 40 are preferably formed from a ¾ inch diameter borosilicate glass rod. Each separation compartment 40 defines a receiving volume of 150 μl. Ion-permeable barriers 18 are preferably isoelectric membranes installed in sealing means 12 including circular silicone pouches 19 that completely prevent liquid loss by wicking. Such an assembled apparatus 10 can be used for the separation of low molecular weight pI markers and proteins and for UV-active carrier ampholyte-based membrane characterization.
In another exemplary embodiment of an electrophoresis apparatus 10, housing means 1 is preferably constructed from LEXAN® and six separation compartments 40 are preferably formed from rectangular ½×¼×1 inch, nonporous, 99.8% alumina blocks. Each separation compartment 40 defines a second dimension b of about 5 mm. Such an assembled apparatus 10 can be used for IET desalting, the separation of low molecular weight pI markers and proteins and for UV-active carrier ampholyte-based membrane characterization. Using a surface treatment with a hydrophilic polymer on sealing means 12 can further reduce leaking problems and practically eliminate electroosmotic flow.
In another exemplary embodiment of an electrophoresis apparatus 10, housing means 1 is preferably constructed from LEXAN® and ten separation compartments 40 are preferably formed from rectangular, ½×¼×1 inch, nonporous, 99.8% alumina blocks, each having a second dimension b being about 18 mm deep. Such an assembled apparatus 10 can be used for IET desalting, the separation of low molecular weight pI markers and proteins, for UV-active carrier ampholyte-based membrane characterization, and for the selection of the appropriate isoelectric membranes for larger scale membrane-based IET separations.
In yet another exemplary embodiment of an electrophoresis apparatus 10, housing means 1 is preferably constructed from LEXAN® and twenty separation compartments 40 are preferably formed from rectangular, 2×35×55 mm, nonporous alumina blocks, each defining second dimension b as being about 40 mm deep. Such an assembled apparatus 10 can be used for IET desalting, the separation of low molecular weight pI markers and proteins, for UV-active carrier ampholyte-based membrane characterization and for the selection of the appropriate isoelectric membranes for larger scale membrane-based IET separations.
The method of altering a composition of a sample by electrophoresis using an apparatus 10, 10′ or 10″ includes selecting an ion-permeable barrier 18 for use between the anode and cathode compartments based upon the given application. Upon providing anode and cathode compartment 14, 15 with the requisite electrolyte, the sample can be added to one or more of compartments 14, 15 and 40. Alternatively, using an apparatus 10, 10′ or 10″, a sample can be added to one or more compartments 14, 15, or 40 and an electrolyte can be added to any compartment 14, 15, or 40 that does not contain the sample. Alternatively, using apparatus 10, 10′, or 10″ both a sample and an electrolyte can be added to one or more of compartments 14, 15, or 40 and an electrolyte can be added to any compartment 14, 15, or 40 that does not contain the sample. Subsequently, an electrophoretic direct current between the anode and the cathode can be provided by applying an electric potential between the anode and the cathode so as to cause a part of a component of the sample being processed to transfer across an ion-permeable barrier 18.
In a first preferred method of processing a sample using an electrophoresis apparatus 10, 10′ or 10″, all ion-permeable barriers 18 inter-disposed in housing means 1 are preferably anti-convective isoelectric barriers. Selecting ion-permeable barriers 18 of this type produces fractions with predetermined pI ranges, i.e., the system is operated in pure IET mode. The pI cuts can be as narrow or as broad as desired, depending on the characteristics of the sample and the objective of the electrophoretic separation, i.e., prefractionation, selective component removal and/or enrichment of a component of the sample being processed. Fractionation can be achieved in the presence or absence of carrier ampholytes and auxiliary isoelectric buffers. This method of processing is especially flexible when compartments 14, 15 and 40 are variable within a single apparatus 10, 10′, 10″ with respect to first characteristic dimension a.
In the second or alternative method of processing a sample using an electrophoresis apparatus 10, 10′ or 10″, ion-permeable barriers 18 adjacent to anode compartment 14 and cathode compartment 15 are preferably anti-convective, isoelectric barriers. All other ion-permeable barriers 18 of apparatus 10, 10′ are preferably anti-convective, ion-permeable, non-isoelectric membranes. The fractions produced in the anode, cathode and/or separation compartments 14, 15, 40 still have distinct pI ranges. However, the respective pI ranges are not known a-priori, rather they depend on the composition of the solution, i.e., the relative amount of the carrier ampholytes, if used, the isoelectric auxiliary agent(s), if used, and the analytes (pure autofocusing mode). The advantage of this method or processing is that it allows for the production of fractions with pI ranges for which no isoelectric membranes are available. The drawback of this second method can be that the pI range boundaries associated with individual compartments 40 cannot be defined by the user ahead of the time. This second method of processing a sample is especially flexible when apparatus 10, 10′ includes a large number of separation compartments 40, each with a very small third characteristic dimension c.
In another or third method of processing a sample using an electrophoresis apparatus 10 having at least two separation compartments 40, ion-permeable barriers 18 adjacent to anode compartment 14 and cathode compartment 15 are preferably anti-convective isoelectric barriers, at least one ion-permeable barrier 18 inter-disposed between separation compartments 40 is preferably an anti-convective, isoelectric barrier, and at least one other ion-permeable barrier 18 is preferably an anti-convective, non-isoelectric barrier. Using this alternative method, the fractions produced in anode, cathode and/or separation compartments 14, 15, 40 also have distinct pI ranges: for some of them the pI range depends on the pI values of the isoelectric membranes delimiting the individual separation compartments 40, for others it depends on the composition of the solution, i.e., the relative amount of the carrier ampholytes, if used, the isoelectric auxiliary agent(s), if used, and the analytes (mixed IET—autofocusing mode). This third or alternative method of processing a sample is advantageous when the pI boundaries for a major sample component are not known exactly, but one still would like to isolate minor components with slightly lower and slightly higher pI values than the pI value of a major component. The drawback of the method is that the exact pI range boundaries of all the fractions cannot be defined by the user ahead of time. The third operation mode also benefits from the use of a relatively large number of separation compartments 40 having a very small third characteristic dimension c.
In yet another or fourth method of processing a sample using an electrophoresis apparatus 10, 10′, ion-permeable barriers 18 adjacent to anode compartment 14 and cathode compartment 15 are preferably anti-convective, isoelectric barriers. The solutions in anode, cathode and separation compartments 14, 15, 40 can contain one or more isoelectric auxiliary agents. Additionally, at least one of ion-permeable barriers 18 of apparatus 10, 10′ is preferably an anti-convective barriers having a characteristic, size-dependent permeability. This alternative method of processing a sample allows for a size-based fractionation of components, especially when the amounts of sample components are relatively small compared to that of the isoelectric auxiliary agent(s) retained in the system by isoelectric trapping.
In another alternative or fifth method of processing a sample using an electrophoresis apparatus 10, 10′, 10″ all ion-permeable barriers 18 are anti-convective and have a characteristic size-dependent permeability. At least one of anode, cathode and separation compartments 14, 15 and 40 can contain a solution of one or more isoelectric auxiliary agents. This method can be used for a rapid desalting of the sample or a size-based or charge-sign-based separation of its components. In a preferred method of desalting using the fifth method of processing a sample in apparatus 10, 10′, the smaller the number of separation compartments 40 provided, the faster the desalting, though the use of at least one separation compartment 40 adjacent to each of anode and cathode compartments 14, 15 might reduce the extent of protic shock for the sample components.
In yet another alternative or sixth method of processing a sample using an electrophoresis apparatus 10, (known as a matrix deployment method), a plurality of separation compartments 40 and inter-disposed isoelectric ion-permeable barriers 18 ranging between a low and a high pI are provided, e.g., twelve separation compartments 40 and ten inter-disposed isoelectric ion-permeable barriers 18 ranging between a pI of 2 to a pI of 12 are provided, where the pI of each successive ion-permeable barrier 18 increases by 1.0. A complex biological sample, for example, a sample intended for proteomic analysis, is loaded into one or more of the ten separation compartments of first apparatus 10. Anode and cathode compartments 14, 15 of first apparatus 10 are filled with an anolyte and catholyte, respectively. In this preferred method of use of electrophoresis apparatus 10, the fractions produced in compartments 40 define the ten rows of a separation matrix.
After performing an IET separation for 10 to 30 minutes using first apparatus 10, the content of each separation compartment 40 is transferred, preferably simultaneously, into ten separate apparatuses 10, each having an anode compartment, a cathode compartment and ten separation compartments 40. The ten apparatuses 10 in the second set of apparatuses define ten columns of the separation matrix. Accordingly, separation compartments 40 present in this second set of apparatuses 10 define the elements of the separation matrix. Ion-permeable barriers 18 adjacent to anode and cathode compartments 14, 15 in apparatus 10 defining the columns of the separation matrix have the same pI values as ion-permeable barriers 18 inter-disposed between the respective separation compartments 40 of first apparatus 10 defining the rows of the separation matrix. Thus, e.g., ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the first column of the separation matrix has a pI of 2, and ion-permeable barrier 18 between the cathode compartment and the last separation compartment of apparatus 10 defining the first column of the separation matrix has a pI of 3; ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the second column of the separation matrix has a pI of 3, and ion-permeable barrier 18 between the cathode compartment and the last separation compartment of apparatus 10 defining the second column of the separation matrix has a pI of 4; ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the third column of the separation matrix has a pI of 4, and ion-permeable barrier 18 between the cathode compartment and the first separation compartment of apparatus 10 defining the third column of the separation matrix has a pI of 5; etc. In each apparatus 10 defining the columns of the separation matrix, separation compartments 40 are isolated from each other by anti-convective, non-isoelectric ion-permeable barriers 18. Thus, a temporally stable pH gradient is formed during the second electrophoretic separation across separation compartments 40 in each apparatus 10 defining the columns of the separation matrix, with the shape of the respective pH gradients depending on the relative amounts of the carrier ampholytes, where used, the auxiliary isoelectric buffers, where used, and the sample constituents. Thus, separation compartments 40 in the first and second sets of apparatuses 10 define the elements of the separation matrix (10×10=100), and each respective separation compartment 40 contains fractions with a pI range of about 0.1. The resulting fractions can then be directly analyzed by mass spectroscopy, used for further research or digested and analyzed by mass spectrometry as common in proteomics to identify the constituent proteins.
If needed, the fractions can be subdivided further, preferably in another electrophoresis apparatus 10 wherein ion-permeable barriers 18 having a characteristic, size-dependent permeability are used in a manner substantially similar to the sixth method of processing a sample as described above. The resulting fractions can then be directly analyzed by mass spectroscopy, used for further research or digested and analyzed by mass spectrometry as common in proteomics to identify the constituent proteins. If needed, the respective digests can also be subjected to a subsequent IET separation in another apparatus 10 to provide fractions containing peptides with similar acidities. Such fractions are preferred for mass spectrometric analysis. This matrix operation mode provides a purely liquid-vein alternative 2DE-MS method for an analysis of the constituents of a complex, proteomic sample.
In an alternative method to the matrix deployment method, the content of each separation compartment 40 from the first IET separation is first stored, then sequentially transferred, ten times, into the ten separation compartments (wells) of the same, sequentially used electrophoresis apparatus 10, and the IET analysis defining the columns of the separation matrix is accomplished over a longer period of time, requiring only a single apparatus 10.
In yet another alternative embodiment of the matrix deployment method described above, a first apparatus 10 having twenty-two separation compartments 40 is assembled using twenty inter-disposed isoelectric ion-permeable barriers 18 having pI values ranging between a pI of 2 to a pI of 12, where the pI of each successive ion-permeable barrier 18 increases by 0.5. A complex biological sample, for example, a sample intended for proteomic analysis, is loaded into one or more of the twenty separation compartments of first apparatus 10. Anode and cathode compartments 14, 15 of first apparatus 10 are filled with an anolyte and catholyte, respectively. In this preferred method of use of electrophoresis apparatus 10, the fractions produced in compartments 40 define the twenty rows of the separation matrix.
After performing an IET separation for 10 to 30 minutes using first apparatus 10, the content of each separation compartment 40 is transferred, preferably simultaneously, into twenty separate apparatuses 10, each having an anode compartment, a cathode compartment and twenty separation compartments 40. This second set of electrophoretic devices, comprised of twenty apparatuses 10, defines the columns of the separation matrix. Accordingly, separation compartments 40 present in this second set of apparatuses 10 define the elements of the separation matrix. Ion-permeable barriers 18 adjacent to anode and cathode compartments 14, 15 in apparatus 10 defining the columns of the separation matrix have the same pI values as ion-permeable barriers 18 inter-disposed between the respective separation compartments of first apparatus 10 defining the rows of the separation matrix. Thus, e.g., ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the first column of the separation matrix has a pI of 2, and ion-permeable barrier 18 between the cathode compartment and the last separation compartment of apparatus 10 defining the first column of the separation matrix has a pI of 2.5; ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the second column of the separation matrix has a pI of 2.5, and ion-permeable barrier 18 between the cathode compartment and the last separation compartment of apparatus 10 defining the second column of the separation matrix has a pI of 3; ion-permeable barrier 18 between the anode compartment and the first separation compartment of apparatus 10 defining the third column of the separation matrix has a pI of 3.0, and ion-permeable barrier 18 between the cathode compartment and the first separation compartment of apparatus 10 defining the third column of the separation matrix has a pI of 3.5; etc. In each apparatus 10 defining the columns of the separation matrix, separation compartments 40 are isolated from each other by anti-convective, non-isoelectric ion-permeable barriers 18. Thus, a temporally stable pH gradient is formed during the second electrophoretic separation across separation compartments 40 in each apparatus 10 defining the columns of the separation matrix, with the shape of the respective pH gradients depending on the relative amounts of the carrier ampholytes, where used, the auxiliary isoelectric buffers, where used, and the sample constituents. Thus, separation compartments 40 in the first and second sets of apparatuses 10 define the elements of the separation matrix (20×20=400), and each respective separation compartment 40 contains fractions with a pI range of about 0.025. The resulting fractions can then be directly analyzed by mass spectroscopy, used for further research or digested and analyzed by mass spectrometry as common in proteomics to identify the constituent proteins.
If needed, the fractions can be subdivided further, preferably in another electrophoresis apparatus 10 wherein ion-permeable barriers 18 having a characteristic, size-dependent permeability are used in a manner substantially similar to the method of processing a sample as described above. Due to the fine pI resolution, the number of size-based fractions required might be relatively low (e.g., 4 to 6). The resulting fractions can then be directly analyzed by mass spectroscopy, used for further research or digested and analyzed by mass spectrometry as common in proteomics to identify the constituent proteins. If needed, the respective digests can also be subjected to a subsequent IET separation in another apparatus 10 to provide fractions containing peptides with similar acidities. Such fractions are preferred for mass spectrometric analysis. This high resolution matrix operation mode can provide a purely liquid-vein alternative to the 2DE-MS analysis of the constituents of a complex, proteomic sample and is believed to be just as (or more) powerful as the currently used prefractionation-2DE-MS methods, while being more suitable for robotics-based automation.
Another or seventh method of processing a sample includes using an electrophoresis apparatus 10 in which dilute samples or fractions can be concentrated by IET. A preferred apparatus 10 is assembled using separation compartments 40, more specifically, a first separation compartment 22 and at least a second separation compartment 23 smaller that the first separation compartment 22, each compartment disposed between anode compartment 14 and cathode compartment 15. Larger separation compartment 22 is preferably located adjacent to anode or cathode compartment 14, 15 (or both, if two larger separation compartments 22 are used). Preferably, anode and cathode compartments 14, 15 are each relatively large as compared to first and at least second compartments 22, 23. Walls 37, 42 of first separation compartment 22 are preferably tapered, producing a smooth transition between anode and cathode compartments 14, 15 having preferably wider first dimensions a and second separation compartment 23 having preferably narrower first dimension a. To provide adequate potential drop across separation compartments 22, 23, at least one isoelectric buffer is added to the sample to be fractionated. The pI value of the added isoelectric buffer is selected such that the isoelectric buffer is trapped in first separation compartment 22, between isoelectric ion-permeable barriers 18 separating anode and cathode compartments 14, 15 and first separation compartment 22, and isoelectric ion-permeable barrier 18 separating the large volume wells and at least one second smaller separation compartment 23. In another preferred embodiment, simultaneous concentration and fractionation can be achieved using a plurality of separation compartments 40 separated by isoelectric or non-isoelectric ion-permeable barriers 18.
An electrophoresis apparatus was assembled using six alumina elements that each contain a 40×2×2.5 mm compartment. The anode, cathode and four separation compartments were separated by five ion-permeable barriers made from isoelectric membranes respectively having pI values of: pI=2, pI=3, pI=5, pI=6.5, and pI=9.5. The anode compartment was filled with 60 mM methanesulfonic acid, and the cathode compartment was filled with a mixture of 20 mM lysine and 20 mM arginine. The separation compartment delimited by ion-permeable barriers of pI=2 and pI=3 contained 50 mM IDA. Nominal 200 μl aliquots of a sample containing 2% Pharmalyte 3<pI<10 carrier ampholytes and three pI markers: nicotinic acid (pI=3.2), 4-hydroxy-2-(morpholinomethylene)-benzoic acid (pI=5.8) and epinephrine (pI=9.2) were loaded into each of the separation compartments of the apparatus. The power supply was operated at a constant power of 4 W for 14 min, yielding an initial potential of 213 V, final potential of 575 V, initial current of 16 mA and final current of 7 mA. The separation took a total of 121 Vh.
The content of each well was analyzed by the iCE280 ICIEF system (Convergent Bioscience, Toronto, Canada) before the IET separation and after the IET separation. The respective volume changes, the component peak areas and their ratios are set out in Table 1.
The results of an ICIEF run are shown in
An electrophoresis apparatus was assembled using four alumina elements that each contain a 40×2×2.5 mm separation compartment. The anode, cathode and two separation compartments were isolated by three ion-permeable barriers made from isoelectric membranes, the first of which had a pI value of 2, the second one was the membrane to be tested, and the third one was a membrane with a pI value of 11.5. The anode compartment was filled with 60 mM methanesulfonic acid and the cathode compartment was filled with 60 mM NaOH. Nominal 200 μl aliquots of a sample containing 2% Pharmalyte 3<pI<10 carrier ampholytes and 0.1% UV active carrier ampholytes were loaded into the two separation compartments. The power supply was operated at a constant power of 6 W for 15 min. After IET, the contents of the well adjacent to the anode compartment and the cathode compartment were analyzed by ICIEF using the iCE280 unit. The results are shown in
An electrophoresis apparatus was assembled using five alumina elements that each contain a 40×2×2.5 mm alumina compartment. The anode, cathode and three separation compartments were isolated by ion-permeable barriers made from isoelectric membranes respectively having pI values of: pI=4; pI=5.6, pI=8.5 and a pI=12. The anode compartment was filled with 50 mM IDA and the cathode compartment was filled with 60 mM NaOH. Nominal 200 μl aliquots of filtered egg white dissolved in 2% Pharmalyte 3<pI<10 carrier ampholytes were loaded into each of the three separation compartments. The power supply was operated at a constant potential of 500 V for 18 min, yielding a final current of 4 mA. After IET separation, the content of each compartment was analyzed by the iCE280 ICIEF system.
The results are shown in
An electrophoresis apparatus was assembled using four alumina elements that each contain a 40×2×2.5 mm compartment. The anode, cathode and two separation compartments were separated by three ion-permeable barriers respectively having pI values of: pI=5, pI=6.5 and pI=9.5. The anode compartment was filled with 60 mM CH3SO3H and the cathode compartment was filled with a mixture of 20 mM lysine and 20 mM arginine. Nominal 200 μl aliquots of 0.5 mg/ml calf liver lysate (7 M urea, 2 M thiourea, 4% CHAPS, 3% 3<pI<10 Pharmalyte carrier ampholytes) were loaded into each of the three separation compartments. The power supply was operated at a constant power of 4 W for 15 min, yielding a final current of 5 mA. After IET separation, the content of each compartment was analyzed by IEF using 3<pI<10 IEF gels (Invitrogen). Results of the separation are shown in
An electrophoresis apparatus was assembled using five alumina elements that each contain a 40×2×2.5 mm compartment. The anode, cathode and three separation compartments were separated by four ion-permeable barriers made from isoelectric membranes respectively having pI values of: pI=3, pI=5, pI=6.5 and pI=9.5. The anode compartment was filled with 60 mM CH3SO3H and the cathode compartment was filled with a mixture of 20 mM lysine and 20 mM arginine. Nominal 200 μl aliquots of 0.5 mg/ml calf liver lysate (7 M urea, 2 M thiourea, 4% CHAPS, 3% 3<pI<10 Pharmalyte carrier ampholytes) were loaded into a single separation compartment delimited by ion-permeable barriers of pI=3 and pI=5. The power supply was operated at a constant power of 4 W for 15 min, yielding a final current of 5 mA. After IET separation, the content of each well was analyzed by IEF using 3<pI<10 IEF gels (Invitrogen). Results of the separation are shown in
The electrophoresis apparatus described herein addresses many of the disadvantages of currently used isoelectric pre-fractionation apparatuses and methods such as the inability to tolerate high electric power loads, the need for active cooling, slow separation speeds, inconvenient system set-up and sample handling, and relatively large sample volumes that cannot be varied easily. The apparatus described herein may be used to separate varying volumes of complex samples into multiple fractions, with direct recovery of the fractions for subsequent analytical or biological characterization, in 10 to 30 min, using 5 to 10 W power, without active (forced) external cooling.
While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims, and equivalents thereof.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/35466 | 10/25/2004 | WO | 1/8/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60513533 | Oct 2003 | US |
