WO2006036119A1

WO2006036119A1 - Method and device for isoelectric focusing

Info

Publication number: WO2006036119A1
Application number: PCT/SE2005/001427
Authority: WO
Inventors: Bengt Bjellqvist
Original assignee: Ge Healthcare Bio-Sciences Ab
Priority date: 2004-09-30
Filing date: 2005-09-28
Publication date: 2006-04-06
Also published as: SE0402375D0

Abstract

The present invention relates to the field of electrophoresis and more specifically to a method and device of separating protein and/or peptide components by isoelectric focusing by arranging the gels or strips, preferably IPG (immobilised pH gradient) gel(s) or strip(s), in a novel way during focusing. The gels or strips are arranged with the width of the gels in substantially vertical direction. This arrangement occupies a minimum of space for long iPG gels and avoids liquid spreading on the gel during long focusing times. IEF run in the device of the invention gives high protein resolution and enables analysis of low abundant proteins.

Description

METHOD AND DEVICE FOR ISOELECTRIC FOCUSING

Technical field

The present invention relates to the field of electrophoresis and more specifically to a method and device of separating protein and/or peptide components by isoelectric focusing by arranging the gels or strips, preferably IPG (immobilised pH gradient) gels or strips, in a novel way during focusing. The gels or strips are arranged with the width of the gels in substantially vertical direction.

Background

The isolation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein-based pharmaceutical compounds. Similarly there is also a need to separate biomolecules for analytical purposes in order to be able to quantitate and identify the proteins and/or peptides present in a sample. Electrophoretic methods are commonly used in the separation step. A wide variety of methods are used for the detection and quantification of the separated proteins. For identification and characterisation of separated proteins MS methods are normally used as these methods are fast and require very small amounts of proteins and/or peptides.

In general terms, electrophoresis involves the movement of charged particles or ions in an electric field. The driving force for the electrophoretic transport of an ion or a particle is the product of the effective charge of the particle and the potential gradient, and the frictional resistance of the medium balances this force.

Based on how the pH and ionic strength is established along the separation distance, basically three different types of electrophoretic methods can be distinguished: zone electrophoresis, isoelectric focusing and isotachophoresis.

In isoelectric focusing (IEF), the separation takes place in a stationary pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways: with the aid of a solution of carrier ampholytes or with an immobilised pH gradient.

In the case of an immobilised pH gradient (IPG) the charged or chargeable groups generating the pH gradient is bound either to the wall of a capillary system or to the matrix when some kind of gel is used to get convection stabilisation. The immobilised charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated. The concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode. A commercially available example of a system for generation of immobilised pH gradients is the lmmobiline Il system™ (Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed. Immobilised pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilised gradient determine the resulting pH gradient, while the carrier ampholytes contribute with conductivity.

The length of the application zone is not critical in isoelectric focusing. In principle, the sample can be mixed into the separation medium and at the start of the separations be present all along the separation distance, but for analytical applications the sample is normally applied close to either the anode or the cathode.

If small sample volumes are used, they are usually applied in sample cups but when larger volumes are used the sample is included in the solution used for rehydration of the IPG strip and/or added to large filter paper positioned between the end of the IPG- strip and either the anodic or cathodic electrode so called "paper bridge" application (Sabounchi-Schutt F, Astrom J, Olsson I, Eklund A, Grunewald J, Bjellqvist B. An immobiline DryStrip application method enabling high-capacity two-dimensional gel electrophoresis.Electrophoresis. 2000 Nov;21(17):3649-56).

To provide the convectional stabilisation, capillaries and different types of gels are used also in isoelectric focusing . Examples of wet gels ready to use are Ampholine PAGplate™ gels, which exist for a number of pH ranges pH3.5-9.5, pH 4.0-6.5, pH 5.5- 8.5 and pH 4.0-5.0. Examples of dry gels are Clen Gel IEF™ and Immobiline Dry Plate™ gels. A special variant of the latter type is the lmmobiline DryStrip™ gels, which are designed to be used as first dimension in two-dimensional electrophoresis.

Commercial products of the last type are normally cast on a plastic foil as 0.5 mm thick gels with a height of 7-24 cm. After polymerisation the gels are washed and dried and then cut into 3.0-3.5 mm wide strips. Prior to use, these strips are rehydrated to acquire a thickness of roughly 0.5 mm. It is important to control the volume increase of the strips in the rehydration step, which is done either through the amount of liquid added to the strip or by physical constrains such as rehydrating the strips between glass- plates limiting the thickness of the rehydrated strip to 0.5 mm. Excessive rehydration to a thickness larger than the original will cause liquid exudation during focusing. The exuded liquid originally contains protein and if the liquid remains in contact with the strip additional proteins will diffuse from the strip out to this liquid. The spreading of the liquid along the strip will result also in protein transport and 2-D maps showing extensive horisontal streaking. Streaking connected to liquid exudation can also be caused by electrosmosis generated by the transport of hydrogen ions, hydroxyl ions, buffer ions or proteins in a strip containing an immobilised pH gradients. In an lmmobiline DryStrip™ containing a pH gradient pH 4-5 the presence of hydrogen ions transported towards the cathode will cause electrosmotic pumping of liquid towards the cathode and exudation of liquid at or close to the cathode. A filter paper placed between the electrode and the IPG-strip is normally used to absorb exuded liquid. If the sample added to the strip contains large amounts of ions transported to the cathode or if very long focusing times are required spreading of this liquid along the IPG strip can still not be avoided unless the paper is exchanged a couple of times during the focusing. If the sample contains large amount of an amphoteric substances, such as glycine or other amino acids or alternatively a Good^'s buffers like MOPS or HEPES which focus within the pH range covered by the IPG strip, water will accumulated at the isoelectric point and exudation and streaking will take place and the same thing will happen if the sample contains large amounts (in the region 10 to 100 ug) of an individual protein focusing within the pH region.

Generally, band sharpness improves with an increase of focusing time and voltage, but this improvement is frequently counteracted by the fact that with an increase of voltage and focusing time, liquid exudation and the connected streaking appears at lower loads of amphoteric substances like proteins, peptides or amino acids. Presently IPG strips are focused with the use of one of the following geometries:

1/ IPG strips are placed on a flat horizontal surface with the plastic foil in contact with a cooled bottom surface.

2/ IPG strips are placed on a flat horizontal surface with the gel side in contact with a cooled bottom surface, such as in US 6 113 766 (IPGphor).

3/ IPG strips are allowed to hang down though anoil as taught by Large Scale Biology.

4/ IPG strips are according to WO 02/ 070111 placed on the surface of a rotating drum.

With the geometries 1 and 2 exuded liquid will spread along the IPG strip with the aid of capillary forces. With the geometries 3 and 4 exuded liquid will drain off the strip in the direction of the pH gradient and for all the geometries the result of exuded liquid will be horizontal streaking in the resulting 2-D maps.

The IPG strips contain preformed pH gradients covering different pH intervals, such as 3-5-6, 5.3-6.5, 6.2-7.5, 7-11 , but also in broader intervals like pH 3-11. The resolution resulting in an isoelectric focusing experiment increases with decreased pH gradient slope, dpH/dx. In many cases the resolution given with a broad gradient, such as 3-10 in a 24 cm long IPG strip, is inadequate and focusing in narrow pH range strips is required. A problem appearing in this context is that very frequently the sample amounts available are limited and not large enough to allow sample application on 3-4 narrow range strips. One solution to limited sample amounts is to make a prefractionation with broad isoelectric focusing in order to split the sample in fractions containing proteins with isoelectric points in narrow pH intervals such as 4-5, 5-6 and so on suitable to be used for sample application on corresponding narrow range IPG- strips. Two different approaches of this type are described in Zuo X, Speicher DW. Comprehensive analysis of complex proteomes using microscale solution isoelectro- focusing prior to narrow pH range two-dimensional electrophoresis. Proteomics. 2002 Jan; 2(1 ):58-68 and in Gδrg A, Boguth G, Kopf A, Reil G, Parlar H, Weiss W. Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two- dimensional gels. Proteomics. 2002 Dec; 2(12):1652-7. However, focusing in two steps is complicated and time consuming. DE 10307907 proposes another solution to the problem with low sample amounts, namely the use of long IPG gels with broad pH intervals. Here a long IPG gei is provided being at least 30 cm, preferably 100 cm or more. The IPG gel is produced under controlled conditions. The IEF is run with a high voltage (10 000V- 30 000V) and an electrically insulating oil is used that surrounds the electrodes and the IPG gel during the run. This long IPG gel is spacious and to avoid that exuded liquid spreads along the gel strip during the long IEF run repeated exchanges of electrode filter papers at the anodic and cathodic electrode is required during focusing.

WO 02/ 070111 also describes a method for manufacture of long IPG gels. In the method of separating a protein mixture by said IPG gel, the gel is placed on a rotating drum. This technique avoids some of the above drawbacks with long IPG gels but is complicated and needs special equipment.

None of the IPG gels within prior art are run with the width of the gel arranged against a substantially vertical surface.

Summary of the invention

The present invention provides a method of running isoelectric focusing which avoids liquid exudation resulting in spreading of protein along the gel strip within which the focusing is done. Furthermore, the invention provides a simplified way of running long gels or strips with high resolution. The method enables focusing of proteins and peptides in sharp spots.

The invention also provides a device for use in said method. The device should be adjusted for IEF with both short and long strips or gels. Preferably, the device should be designed to avoid liquid exudation spreading in the direction of the pH gradient on the gel or strip. For high voltage applications, the liquid exuded should not be allowed to be in contact with the gel.

Thus, in a first aspect the invention relates to a method of separating proteins/peptides in a biological mixture by isoelectric focusing (IEF) using gel(s) or strips, preferably immobilised pH gradient (IPG) gel(s) or strip(s), comprising the following steps: a) placing the gel(s) in channel(s) of a focusing unit with the width of the gel(s) arranged substantially vertically in said channel(s) and applying sample to the gel(s) either before and/or after placing the gels in the focusing unit; b) positioning of electrodes on said gel(s), wherein a cathode is placed at the basic end and an anode at the acidic end of each of said gel(s); and c) applying a voltage between said cathode(s) and anode(s), wherein, during focusing, any exudates formed from the gel(s) is drained off from the gel.

This method may be run will IPG gels of any length, such as 7-24 cm or longer. Substantially vertical means in this context that it is substantially perpendicular to the direction of focusing, i.e. to the direction of the pH-gradient of the IPG gel.

Preferably, a low viscosity paraffin or silicone oil is added to cover the gel(s) and electrodes after step b). If no oil is added, the method may be run in a closed system. Oil or a closed system is needed because of the high voltages applied but also as a mean to avoid evaporation of water and crystallisation of urea in the IPG strips.

In a preferred embodiment, the IPG gel is at least 30 cm long, preferably at least 50 cm long and more preferably at least 90 cm long, preferably 96 cm.

If the gel is 96 cm it can be divided into 4 x 24 cm after the run and this length is adopted to the second dimension of 2D electrophoresis.

The gel or strip may also be divided into two or more sections before IEF which are connected to each other, via for example polyacrylamide bridges as described in WO 05/026715. In this way, serial IEF may be run.

Preferably, the channels are curve shaped. For long IPG gels, such as over 30 cm, the channels are spiral formed. The pH-gradient of the IPG gel is optional. Especially for long IPG gels, a broad pH range will be used, such as pH 3-10 or 3-11 or 3-12.

The gel(s) or strip(s) may be rehydrated before or after it/they is/are placed in said channel(s). In a preferred embodiment, during IEF, excess liquid is allowed to drain off the gel(s) into the bottom of the channel(s) in such a way that the gel(s) no longer is/are in contact with said liquid. This is a great advantage of the invention since no excess liquid from the gel will interfere with and disturb the separation process.

Long IPG strips have the capability of resolving proteins in a broad pH range due to the simple fact that they are long and thereby the distance between focused spots with different pH becomes larger. Preferably, a organic disulfide such as DeStreak™ is present in the rehydration solution.

DeStreak™ prevents streaking of the proteins and peptides and thereby gives more distincly focused spots.

In the method according to the invention at least two IPG gels may be placed in parallel but insulated from each other in separate channels in the focusing unit.

In a second aspect, the invention relates to a device for use in the above method comprising a focusing unit (1) provided with curve-shaped channel(s) (2) and electrodes (3).

Preferably, the channels are spiral formed and have a length of at least 30 cm. Preferably, the channels have a width of 1-10 mm, preferably 3-6 mm, and a depth of 7-15 mm.

Thus, the device is adjusted for IEF running of long IPG strips or gels by arranging the strips or gels spirally in the device with the gel width in a substantially vertical direction.

In a preferred embodiment, a ridge or shelf is provided in the channels to prevent the gels from sliding down to the bottom of said channels and to allow liquid to drain off to a bottom space in the channels. The ridge or shelf may be divided into separate sections and may be in the form of distance laths. Preferably, at least two channels are arranged in parallel in said focusing unit. Preferably, 1-6 gels can be arranged in 1-6 channels in the device. The number of electrode pairs (anode and cathode) in the device corresponds to the number of gels to be run. The outer electrode(s) should preferably be movable for adjustment to gel(s) of different length. The inner electrode(s) may be permanently arranged.

The device according to the invention may be a separate unit or may be integrated in a multipurpose unit, like a Multiphor™, wherein said focusing unit is provided with an inlet and an outlet for a cooling liquid under and/or between the channels. A lid may be arranged over the focusing unit.

Thus, the device of the invention comprises channels in which the gel strips to be focused are placed in horizontal positions and with the plastic foil supporting the gel strip towards one of the vertical walls forming the channel within which the specific strip is placed with the width of the gel substantially in a vertical direction. The channel wall could be straight but also slightly curved representing a part of a wall in a cylinder or tubing.

The channels could be straight but in reality a curved form is preferred as this form make it easier to ascertain good contact between the vertical wall and the film supporting the gel or strip. The channels should contain the arrangements necessary for establishing contact between the two ends of the IPG strips and an anodic and cathodic electrode respectively, and at each end of the strips there should be the space and arrangements required to allow for anodic and/or cathodic paper bridge application. The width of the channels could correspond to the thickness of the rehydrated IPG-strips including the contribution from the support film in which case the IPG strips are pressed (pushed) down towards the bottom of the channel. In this case the channels are possible to use as well for the re-swelling as for the focusing of the IPG strips.

In a preferred form the width of the channels exceeds the thickness of IPG strips in which case a minimum distance of 2 mm is maintained between the bottom of the channels and the IPG strips either with the aid of distance laths or by placing the strip on a shelf where said shelf follows the vertical wall towards which the strip is placed and where the width of said shelf does not exceed the thickness of the IPG strip. The arrangement is made in order to allow exuded liquid to drain off the strip in a direction perpendicular to the pH gradient with minimal spreading along the gel in the direction of the pH gradient and to accumulate in a volume beneath the strip which is large enough to ascertain that there will be no contact between exuded liquid and IPG strip. The minimum depth of the channel should allow the IPG strip to be covered with oil during focusing and result in a minimum distance between the oil-air interface and the IPG strips of 2 mm and preferably this distance should be somewhere between 3 and 6 mm.

In a more preferred form, the above preferred form is provided with curve-shaped or spiral formed channels. These channels optimize the contact between the plastic film and the vertical wall of the channel. The spiral formed channel is especially advantageous for running of long IPG strips since the spiral form requires a minimum of space.

Brief description of the drawings

Fig. 1 shows a schematic top view of device with spiral formed channels for IEF running of long IPG strips.

Fig. 2 is a cross section along line A-A in Fig. 1 and show how the gels and electrodes are arranged in the channels.

Detailed description of the invention

The invention will now be described more closely below in association with the drawings but the invention is not to be construed as limited to the specific embodiments disclosed.

A. Production of long IPG gels

A gel is cast between two glass plates of about 100 cm length, 24 cm breadth and 0.5 mm thickness, wherein the glass plates are sealed along the sides and the bottom. A Gelbond™ or other gel adhering film is tightly arranged on one of the glass plates. A gradient is formed by using a heavy solution of acrylamide/bis acrylamide + 20% glycerol and a light solution of acrylamide/bis acrylamide without glycerol. At the same time a gradient is formed by using a mixure of acid immobilines and basic immobilines, respectively. Thus, the gradient is from 20%-0% glycerol and preferably from pH 3-10, - 11 or -12. In connection with the gradient generation TEMED and ammonium persulphate is added to start the polymerisation reaction.

The gradient starts with only heavy solution and only acid immobilines. The highest concentration of glycerol will be at the bottom of the gel as well as the most acid pH- value. The IPG gel is allowed to polymerise for 1-2 hours. The Gelbond™ films with adhered IPG gels are removed from the glass plates and excess of non-polymerised material is washed off.

Thereafter, the Gelbond™ films with adhered IPG gels are dried in an oven fur sufficient time to dry the gel on the film. Finally, the film is cut into long narrow (0.2-1 cm) strips.

B. Rehydrating IPG gels

It is very important that the IPG strips are rehydrated into a correct size. If they are rehydrated into a size that is too large, too much water will be comprised in the gel and this is not good because during focusing this excess liquid will be squeezed out of the strip together with proteins. If the strips are insufficiently rehydrated, then the time required for focusing will increase due to decreased protein mobilities. Normally the resulting rehydration volume is limited by the amount of liquid added, but it could also be determined through geometrical constraints of the chamber used for the rehydration

The solution used in the rehydration step could have varying composition but normally the solution contains 8-9 M urea or alternatively a mixture of urea and thiourea, some type of non ionic or amphoteric detergent like CHAPS or Triton-X100, carrier ampholyte and either a reducing agent like DTT or an organic disulfide (DeStreak™) . The rehydration is done for a minimum of 6 hours and normally over-night (14-20 hours). To avoid evaporation and urea crystallisation, protection against evaporation is needed. This can be achieved by covering the gel strip with paraffin or silicone oil and/or with a lid enclosing the chamber. The rehydration may be made in the device of the invention in which case the device will be sized for that purpose. C. IEF running in the IPG strips

To allow for a compact flat focusing unit, the strip is placed on its edge (i.e. with its width in a vertical position) for focusing as shown in the cross section of Fig. 2. The strip is spirally arranged in the channel of the device of the invention. Preferably, the strip is rehydrated in the device but may also be rehydrated in a preceding step. The device is placed on a Multiphor™. Sample is added to the strip by either rehydration or paper bridge application. For paper bridge application, a positively charged paper may be used such as described in co-pending application SE 0303581-3.

Anode and cathode electrodes are placed at each end of the strip. Low viscosity paraffin or silicone oil is added to cover the strips, the lid is closed and a voltage is applied. The voltage profile is as common practice, i.e. first a very low voltage to run in the sample and then higher voltages. The vertical positioning of the strips in the channels allows excess liquid formed during the run to flow into the bottom of the channel without contact with the gel. For long IPG gels, the device is built for voltages of 10-10O kV.

As appears in Fig. 2, there is a free space between the bottom of the channel and the bottom gel edge and this is achieved by a ridge or latch positioned in one of the side walls of the channels. Fig. 2 also shows the gel in solid black and the end of an electrode placed in the channels. If the sample is applied by paper bridge application, then a not shown filter paper will be located between the gel and electrode.

The device of the invention offers easy handling and requires a minimum of space for rehydration and IEF running of long IPG gels.

The IEF run may be used as a pre-treatment step before 2D electrophoresis or other separation. The high resolution in the IEF run according to the invention enables analysis of low abundant proteins.

After the IEF run, the proteins in the strips may alternatively be directly further separated in the second electrophoresis step of 2D electrophoresis or may be directly analysed. The strips may be cut into suitable lengths before 2D electrophoresis or other analysis.

Claims

1. A method of separating proteins/peptides in a biological mixture by isoelectric focusing (IEF) using gel(s), comprising the following steps: a) placing the gel(s) in channel(s) of a focusing unit with the width of the gel(s) arranged substantially vertically in said channel(s) and applying sample to the gel(s) either before and/or after placing the gels in the focusing unit; b) positioning of electrodes on said gel(s), wherein a cathode is placed at the basic end and an anode at the acidic end of each of said gel(s); and c) applying a voltage between said cathode(s) and anode(s), wherein, during focusing, any exudates formed from the gel(s) is drained off from the gel.

2. A method according to claim 1 , wherein the gel(s) are immobilised pH gradient (IPG) gel(s) or strip(s).

3. A method according to claim 1 or 2, wherein a low viscosity paraffin or silicone oil is added to cover the gel(s) and electrodes after step b).

4. A method according to claim 1 , 2 or 3, wherein the gel or strip is at least 30 cm long.

5. A method according to claim 4, wherein the gel or strip is at least 50 cm long.

6. A method according to claim 5, wherein the gel or strip is at least 90 cm long,

7. A method according to one or more of the above claims, wherein the gel(s) or strip(s) are divided into two or more sections which are connected to each other.

8. A method according to one or more of claims'! -7, wherein the channels are curve shaped.

9. A method according to one or more of claims1-7, wherein the channels are spiral formed.

10. A method according to claim one or more of the above claims, wherein pH- gradient of the IPG gel is pH 3-11.

11.A method according to one or more of the above claims, wherein said gel is rehydrated before or after it/they is/are placed in said channel(s).

12. A method according to one or more of the above claims, wherein, during IEF, excess liquid is allowed to drain off the gel(s) into the bottom of the channel(s) in such a way that the gel(s) no longer is/are in contact with said liquid.

13. A method according to one or more of the above claims, wherein an organic disulfide is present in the rehydration solution.

14. A method according to one or more of the above claims, wherein at least two IPG gels are placed in parallel but insulated from each other in separate channels.

15. A device for use in a method according to claim 1-14, comprising a focusing unit (1) provided with curve-shaped channel(s) (2) and electrodes (3).

16. A device according to claim 15, wherein the channels (2) are spiral formed.

17. A device according to claim 15 or 16, wherein the channels (2) have a length of at least 30 cm.

18. A device according to claim 15, 16 or 17, wherein the channels (2) have a width of 1-10 mm, preferably 3-6 mm, and a depth of 7-15 mm.

19. A device according to one or more of the claims 15-18, wherein a ridge or latch is provided in the channels to prevent the gels from sliding down to the bottom of said channels.

20. A device according to claim 19, wherein said ridge or latch is divided into separate sections.

21. A device according to one or more of the claims 15-20, wherein at least two channels are arranged in parallel in said focusing unit.

22. A device according to one or more of the claims 15-21 , wherein said focusing unit is provided with an inlet and an outlet for a cooling liquid under and/or between the channels.

23. A device according to one or more of claims 15-22, also comprising a lid to be arranged over the focusing unit.