WO2001094931A2 - Method and apparatus for direct coupling of planar chromatography with mass spectrometry - Google Patents

Method and apparatus for direct coupling of planar chromatography with mass spectrometry Download PDF

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Publication number
WO2001094931A2
WO2001094931A2 PCT/IL2001/000521 IL0100521W WO0194931A2 WO 2001094931 A2 WO2001094931 A2 WO 2001094931A2 IL 0100521 W IL0100521 W IL 0100521W WO 0194931 A2 WO0194931 A2 WO 0194931A2
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Prior art keywords
gel
sampling port
matrix
analytes
apparatus according
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PCT/IL2001/000521
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French (fr)
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WO2001094931A3 (en
Inventor
Ovadia Lev
Genia Gun
Davidovich Alexander Modestov
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
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Priority to US60/209,807 priority
Application filed by Yissum Research Development Company Of The Hebrew University Of Jerusalem filed Critical Yissum Research Development Company Of The Hebrew University Of Jerusalem
Publication of WO2001094931A2 publication Critical patent/WO2001094931A2/en
Publication of WO2001094931A3 publication Critical patent/WO2001094931A3/en

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    • HELECTRICITY
    • H01BASIC ELECTRIC ELEMENTS
    • H01JELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00Particle spectrometers or separator tubes
    • H01J49/02Details
    • H01J49/04Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0431Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416Systems
    • G01N27/447Systems using electrophoresis
    • G01N27/44704Details; Accessories
    • G01N27/44717Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/4473Arrangements for investigating the separated zones, e.g. localising zones by electric means
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/95Detectors specially adapted therefor; Signal analysis

Abstract

A method is disclosed for coupling of planar chromatography and mass spectroscopy, comprising placing a matrix that contains analytes that have been separated by planar chromatography in a gel (2), between at least two electrodes, activating an electrical current (27) in said electrodes, wherein activation of the current induces movement of the analytes out of the matrix, towards one of the electrodes, collecting the analytes from the external surface of the matrix to a sampling port and transferring the analytes to a mass spectrometer (5).

Description

A METHOD FOR DIRECT COUPLING OF PLANAR CHROMATOGRAPHY WITH MASS SPECTROMETRY, AND AN APPARATUS FOR USE THEREOF

FIELD OF THE INVENTION

The present invention relates to a method of coupling and integrating two tools of analytical chemistry: planar chromatography and mass spectroscopy. The method is especially useful for coupling of gel electrophoresis with mass spectrometry. The present invention further relates to an apparatus for use according to the method disclosed herewith.

In the present invention, the method and the apparatus disclosed are applicable for any type of planar chromatography. However, since gel electrophoresis is the most widely used type of planar chromatography, in the present invention, gel electrophoresis is mentioned as a preferred embodiment, not intended to limit the scope of the invention from other types of planar chromatography, such as thin layer chromatography, etc.

In the present invention, the term "matrix" refers to a planar chromatography matrix (such as a silica matrix) or an electrophoresis gel.

In the present invention, the term "materials" used to describe materials that are separated in a planar chromatography matrix, such as an electrophoresis gel, refers to analytes or solutes. The terms "analytes" and "materials" (in this context) are interchangeable, and refer to materials that are undergoing or have underwent separation by planar chromatography.

BACKGROUND OF THE INVENTION

Many disciplines of life science rely on the ability to identify and quantitate macromolecules and dissolved chemical compounds. Morphological taxonomy studies of organisms have been replaced in the degree of importance, by mapping and characterization of nucleic acids and of the appropriate proteins they express. Biopolymers, especially proteins and oligonucleotides, have become the most important biomarkers in medicine and in practically all other fields of life science.

Two central laboratory tools are now used routinely for chemical identification of biochemicals and macromolecules; gel electrophoresis and mass spectrometry (Rabilloud et al., Anal. Chem., Jan. 2000, 48A-55A). Common laboratory procedure for protein or nucleic acid identification involves a first step of gel electrophoresis to separate the biological material on an agarose, a polyacrylamide or another gel. Various types of polyacrylamide gels exist, that vary in the degree of cross-linking and the nature of the surfactant included in the gel; the surfactant having the most widespread use is sodium dodecyl sulfate (SDS), a denaturing anionic surfactant, and the resultant gel and electrophoresis method are termed SDS-PAGE (SDS-polyacrylamide gel electrophoresis).

Most often, a complex mixture of proteins or nucleic acids are first separated on a gel plate in a single direction. Further separation may include electrophoresis in a second dimension. The gel is stained and/or fixated, to view and quantitate the resultant bands, using radioactive labels, fluorescent dyes or colorimetric reagents. Bands of interest can then be excised from the gel, and often must undergo de-staining of florescent or colored dyes, and removal of all traces of the gel, in order to proceed with analysis strategies such as mass spectroscopy, Edman degradation, or HPLC. Excision, de-staining and purification steps entail up to several hours of laboratory work, which lengthens the analysis procedure and make it labor-intensive. These steps often necessitate use of volatile solvents, which are potentially harmful to the researcher and to the environment.

The gel can alternatively be blotted onto a membrane, and Edman degradation or Matrix- Assisted Laser Desorption-Ionization Mass Spectroscopy (MALDI-MS) can be performed directly on the membrane. This eliminates the need for excision of bands, de-staining and purification from the gel.

In MALDI, a laser pulse is used to desorb and ionize the species of interest from a matrix in which it is embedded. The sequence or residues are read directly in the mass spectrometer. The ionization and desorption are followed by Time of Flight (TOF) mass spectroscopy. MALDI-TOF is being more widely applied in biopolymer analysis (Pacolsky & Winograd, 1999, Chem. Rev. 99(10), 2977-3005; Ekstrom et al, 2000, Anal. Chem. 72, 286). However, access to a mass spectrometer of the MALDI-TOF type is not always available, due to the expense of the apparatus. An additional disadvantage associated with MALDI mass spectroscopy is the fact that the results obtained by this method are highly dependant on the type of matrix utilized, and the experimental parameters, making this analysis difficult to repeat and thus, controversial (Zubritsky, 2000, Analyt. Chem., 72, 191A). A further disadvantage of MALDI-MS lies in it causing increased fragmentation of the compound undergoing analysis, which detracts from its protein or nucleic acid identification capabilities. (Recall that biopolymer identification is based on accurate identification of the molecular mass). MALDI-MS is therefore, not always a method of choice for analysis of proteins and nucleic acids, and cannot replace other forms of mass spectroscopy, which require multiple steps of excision and purification from gels.

The need exists for a rapid method of removal of proteins or nucleic acids of interest from a gel, which does not require multiple steps of excision, de-staining and purification from gel remnants or aggressive laser desorption steps.

It is the object of the present invention to provide a method that allows direct coupling of planar chromatography (especially gel electrophoresis) and a mass spectrometer, without the need for excision of bands, de-staining and purification. This method is time-saving and non-labor-intensive. The present invention further provides an automated apparatus for use according to this method.

These and other objects of the present invention will become more apparent from the detailed description of the preferred embodiments, that follows below.

SUMMARY OF THE INVENTION

There is thus provided in the present invention, a method for coupling of planar chromatography or gel electrophoresis and mass spectroscopy, comprising: a) placing a matrix that contains materials that have been separated by planar chromatography or gel electrophoresis in a gel, between at least two electrodes; b) activating an electrical current in said electrodes, wherein activation of the current induces movement of said materials out of the matrix, towards one of said electrodes; c) collecting materials from the surface of said matrix to a sampling port (this transferring is done via the assistance of an electric field) d) transferring the materials to a mass spectrometer.

In accordance with a preferred embodiment of the present invention, the matrix is an electrophoresis gel.

Additionally, in accordance with a preferred embodiment of the present invention, the matrix is essentially comprised of polyacrylamide gel.

Further, in accordance with a preferred embodiment of the present invention, the materials that have been separated are selected from the group consisting of polypeptides, proteins, nucleic acids, amino acids, inorganic polymers, organic polymers or derivatives thereof.

Still further, in accordance with a preferred embodiment of the present invention, the electrodes are located above and below said matrix.

Moreover, in accordance with a preferred embodiment of the present invention, collection of the materials from the surface of the matrix is performed in an automated process.

Additionally, in accordance with a preferred embodiment of the present invention, the transfer of materials to a mass spectrometer is performed in an automated process.

Moreover, in accordance with a preferred embodiment of the present invention, the electrical current activated to transfer the materials from the gel toward the mass spectrometer is sufficient to produce an electrical field between said electrodes of 1- 1000 V/cm.

Further, in accordance with a preferred embodiment of the present invention, the matrix is of a type selected from the group consisting of an agarose gel, or a polyacrylamide gel.

Still further, in accordance with a preferred embodiment of the present invention, the materials separated by planar chromatography in the matrix, have underwent a procedure selected from the group consisting of isoelectric focusing, two-dimensional electrophoresis, or one-dimensional electrophoresis. There is further disclosed in the present invention, an apparatus for coupling of planar chromatography and mass spectroscopy, according to the method defined above, comprising: a) a plate for holding a planar chromatography matrix; b) at least two electrodes; c) a power source for supplying voltage between said electrodes; d) at least one displaceable sampling port, wherein said sampling port is oriented perpendicular to said plate; e) means for producing movement of the sampling port relative to said plate; f) a mass spectrometer and an accompanying computer processor; g) a tubing system attached at one end to said sampling port, wherein said tubing system allows passage of materials from said sampling port to said mass spectrometer.

Further, in accordance with a preferred embodiment of the present invention, the sampling port is a capillary.

Still further, in accordance with a preferred embodiment of the present invention, the sampling port is a porous filter.

Additionally, in accordance with a preferred embodiment of the present invention, one of the electrodes is displaceably located within the planar chromatography matrix.

Moreover, in accordance with a preferred embodiment of the present invention, at least one of the electrodes is displaceably located within the sampling port.

Further, in accordance with a preferred embodiment of the present invention, one of said at least two electrodes is displaceably located adjacent to the sampling port.

Additionally, in accordance with a preferred embodiment of the present invention, the means for producing movement of the sampling port are comprised of computer-controlled robotic arms.

Further, in accordance with a preferred embodiment of the present invention, the apparatus additionally comprises at least one pump and at least one reservoir of auxiliary fluid. The pump is attached to said at least one reservoir and to said tubing system, and said pump is capable of delivering auxiliary fluid from said reservoir into said tubing system. Still further, in accordance with a preferred embodiment of the present invention, the displaceable sampling port contains wet gel.

Additionally, in accordance with a preferred embodiment of the present invention, the displaceable sampling port contains a porous filter in the tip of said sampling port proximal to the matrix plate.

Moreover, in accordance with a preferred embodiment of the present invention, the displaceable sampling port contains a gel plug in the tip of said sampling port proximal to the matrix plate, wherein said gel plug is capable of preventing flow of materials from inside the sampling port to said plate.

Additionally, in accordance with a preferred embodiment of the present invention, the mass spectrometer is selected from the group consisting of an inductive coupled plasma mass spectrometer, an electrospray ionization mass spectrometer, or an atmospheric pressure chemical ionization mass spectrometer.

Further, in accordance with a preferred embodiment of the present invention, the gel plate and the sampling port are enclosed within a pressure chamber.

Still further, in accordance with a preferred embodiment of the present invention, the apparatus additionally comprises a dialysis membrane within the tubing system.

Moreover, in accordance with a preferred embodiment of the present invention, the apparatus additionally comprises a cation exchange membrane within the tubing system.

Additionally, in accordance with a preferred embodiment of the present invention, the apparatus additionally comprises an anion exchange membrane within the tubing system.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is herein described, by way of example only, with reference to the accompanying drawings, wherein:

Figure 1 is a schematic representation of the transfer apparatus of the present invention.

Figure 2 is an isometric view of an apparatus, demonstrating the ability of the capillary to be moved along the surface of the gel to reach bands of interest. Figure 3 A is a schematic illustration of one arrangement of capillaries through which the materials pass to exit from the gel to the mass spectroscope in the apparatus.

Figure 3B is a schematic illustration of a second form of sampling port through which the separated materials pass to exit from the gel to the mass spectrometer.

Figure 4 is a schematic illustration of a sampling port with zero dead volume.

Figure 5 is a schematic illustration of an apparatus having two pipelines for addition of two different auxiliary fluids. This apparatus was used in Examples 1 & 2.

Figure 6 is comprised of four graphs of mass spectral data of four different compounds, recorded during the experiment of Example 1.

Figure 7 is comprised of mass spectral data recorded during the experiment of Example 2.

DETAILED DESCRIPTION OF THE INVENTION

It is appreciated that the detailed description that follows is intended only to illustrate certain preferred embodiments of the present invention. It is in no way intended to limit the scope of the invention, as set out in the claims.

The present invention discloses a method for direct transfer of materials (analytes, molecules and polymers), from an electrophoresis gel, to a mass spectrometer. In the method of the present invention, the charged chemical species of materials of interest in the gel, are made to migrate from the gel in response to an electric field. The materials migrate through a sampling port, which is held in place over the band of interest, to reach a liquid pumped into the area above the sampling port. This area is termed the "elution chamber".

This method of transfer is more rapid and easier than the current practice in the art, which involves excision of bands of interest, followed by purification from gel remnants. The present invention further discloses an apparatus suited for use according to this method.

In the method of the present invention, materials undergoing separation and analysis are submitted to planar separation, for instance to electrophoresis on an agarose or a polyacrylamide gel, according to any appropriate planar chromatography or electrophoresis protocol. (The gel can be, for example, an isoelectrical focusing gel, or a gel where the materials (analytes) were separated in two dimensions, and the substance from which the gel is formed is not limited to agarose or polyacrylamide.) While separation of the analytes (e.g., by electrophoresis) is in progress or after it is completed, a potential is applied between two electrodes, oriented, for instance, so that one is connected to tubing above the gel, and one is below or within the gel. Electrophoresis is then performed in the vertical plane, and the analytes present in the gel, in the area below the sampling port migrate towards the upper electrode, and out of the gel. As the analytes migrate towards the electrode they are collected in a sampling port. The sampling port has an exit that is connected by tubing to a mass spectrometer, and the analytes pass through the tubing into the mass spectrometer.

Referring to Figure 1, an apparatus is depicted, suited for use coupling of gel electrophoresis and mass spectrometry, according to the method proposed above. An electrophoresis gel(2) is placed on a gel plate(13), after electrophoresis has been performed in the classic horizontal direction. A sampling port, in the form of a caρillary(l) is supported by a displaceable robotic arm(14) so that the capillary is oriented in the vertical direction, with one end of the capillary pointing downwards towards the gel. The robotic arm moves the capillary so that it makes contact with the surface of the gel, or is inserted just below the surface of the gel. In this apparatus, an electrode(4) connected to a power source (27) is present within the capillary, and the tip of the electrode extends through the capillary to reach the gel. A wire net (5) is present between the gel and gel plate (13). In other embodiments, the wire net is positioned beneath the gel plat (13) The wire net is connected to the power source(27), and upon activation of the power source, the wire net will act as a second electrode. The electric current is turned on, applying voltage between the electrodes. Since the gel is present between both electrodes, the electrical field causes the analytes (materials) present in the gel at the point directly beneath the capillary(l), to migrate upwards towards the capillary(l), and into said capillary. A supply line(15) opens into the capillary from a side entrance in the capillary, and allows entrance of an auxiliary fluid from an auxiliary pump(16) containing a reservoir of auxiliary fluid. This stream of auxiliary fluid, which can be a solvent such as methanol, accelerates passage of the analytes upwards through the capillary. The materials exit the capillary and pass through tubing termed the "transfer line"(7) to enter a mass spectrometer(ό), where they are analyzed by mass spectroscopy. The transfer line preferably has a minimal volume. The apparatus and the method of the present invention are useful for transferring of polymers from a gel or another planar chromatography matrix, to many types of mass spectrometers, for instance to an inductive coupled plasma mass spectrometer, (ICP-MS), an electrospray ionization mass spectrometer (ESI-MS) or to an atmospheric pressure chemical ionization interfaced mass spectrometer (APCI-MS).

Referring to Figure 2, specific bands of interest(17) may be pre-selected, and the robotic arm(14) pre-programmed so that the sampling port (1) will position itself only above those specific areas. Alternatively, the robotic arms can move and advance the capillary in a set manner over the gel, so that the gel is "scanned" by the capillary, and the electric field is constantly re-applied to a new position on the gel. A computer processor associated with the mass spectrometer(ό) can correlate what area in the gel each sample was removed from, and which mass spectral data are related to that area. In this embodiment, the capillary is moved to different points in the gel by a robotic arm(14) controlled by computer, however, the arm can alternatively be manually positioned and re-positioned. It is also possible to move the gel plate which holds the gel, and keep the sampling port stationary.

The electrodes necessary to generate an electrical field can be positioned at several different areas and can take several different forms:

1) Referring back to Figure 1, the upper electrode can be placed within the tubing(15) supplying the auxiliary fluid, inside the sampling port(4), or in the transfer line tubing(7) which connects the sampling port and the mass spectrometer. One particularly preferred location is in a special fluid-filled chamber(not shown) connected conductively to the tubing that supplies the auxiliary fluid. In this case electrochemical reduction (oxidation) processes which naturally occur at the electrode do not modify or harm the analytes, since the analytes are not in the vicinity of the electrode, and do not contact the electrode.

2) The second electrode can be a miniature (10 micrometer - 1 cm) electrode that can be immersed in the gel in a position close to the sampling port, whereby the electrode and the sampling port are moved together. The electrode can be immersed in a chamber filled with electrolyte, which is connected to the gel plate. It can be located in the gel plate itself, or it can be a metal net or a metal plate beneath the gel.

In order to obtain additional separation of the analytes, the capillary can be as long as 40 cm, and the electrical field applied upon the capillary will further separate the analytes by capillary electrophoresis as the analytes advance upwards through the capillary towards the mass spectrometer. Conventional electrophoresis can thus be performed in a relatively small gel plate, to give only partial separation, and the capillary electrophoresis is a second stage of electrophoresis, which provides additional separation. Though capillary electrophoresis is often followed in the art by mass spectroscopy, the method and apparatus of the present invention differ in that in the present invention, planar chromatography is coupled with mass spectroscopy. The principal separation of analytes is performed in a matrix, as opposed to in a liquid, as in capillary electrophoresis. Addition of a step of capillary electrophoresis is optional in the present invention. Separation in a matrix allows utilization of a variety of advantageous separation techniques not possible during capillary electrophoresis, such as isoelectrical focusing, and two-dimensional electrophoresis.

The tubing connecting the capillary to the mass spectrometer can include a hollow microdialysis membrane for purification of the stream leading to the mass spectrometer.

The main purpose of the sampling port is to allow electrical contact between a particular location on the gel plate and between the auxiliary fluid, which contains electrolytes that surround and dissolve the analytes, and accelerates passage of the analytes to the mass spectrometer. However, any auxiliary fluid that leaks from inside the sampling port to the surface of the electrophoresis gel, and any analytes it contains, will be lost to mass spectrometry analysis, and will not reach the mass spectroscope. Auxiliary fluid can be prevented from returning to the electrophoresis gel by several means:

1) A section of the sampling port can be filled with gel, through which ions can move in response to application of the electrical field, however auxiliary fluid cannot pass through said gel to exit the sampling port. In this case, ions from the planar gel electrophoresis plate pass upwards through the gel/gel interface at the point of contact of the capillary and the gel plate, then go through a section of capillary that is filled by gel, and reach the gel/auxiliary liquid interface.

2) In another preferred embodiment, the flow of analytes from the gel into the capillary is increased by replacing the single capillary with several capillaries. This configutration of the sampling port increases the overall cross-section of the sampling port, yet, should the capillaries be filled with gel, this preserves adhesion of the gel to the capillary walls. It was found that replacing a single gel-filled capillary 0.32mm in diameter, with a single gel-filled capillary having a larger diameter (0.53mm) resulted in poor adhesion of the gel to the inside walls of the capillary. Thus a plurality of small diameter capillaries is preferred. Referring to Figure 3 A, a plurality of gel-filled capillaries(19) receive the materials as they migrate out of the gel(2) in response to the electrical current when the current is applied in the vertical plane. Auxiliary fluid enters from the auxiliary fluid supply line(15), to accelerate passage to the mass spectrometer(ό).

3) Referring to Figure 3B, the bundle of capillaries can be substituted by a porous filter(20). Filling the filter by gel prevents bulk flow of liquid through it.

4) Referring to Figure 4, in this embodiment leakage of auxiliary liquid to the gel plate is prevented by placing a plug(22) of a narrow pore filter in a casing(34) on the sampling port. The plug is pressed to the planar chromatography gel, preventing flow of auxiliary liquid between the walls of the casing and the gel. Often when a hollow capillary is inserted into and moved within a gel, fracturing of the gel occurs. The narrow pore filter(22) prevents pieces of fractured planar chromatography gel from entering the sampling port and blocking the transfer line. In the diagram shown in Figure 4, the narrow pore filter holds auxiliary fluid above the filter within the sampling port, and as the analytes migrate electrophoretically upwards, they leave the gel and reach the auxiliary fluid above the filter.

Referring back to Figures 3 A and 3B, an apparent drawback of the sampling ports shown in Figures 3 A and 3B is the relatively large volume of the elution chamber(35) located above the capillary in Figure 3A, or above the filter in Figure 3B. The volume of the elution chamber is a "dead volume" of the system, which increases the response time and thus decreases the sensitivity of the apparatus.

Referring to Figure 4, a sampling port configuration is shown, having a dead volume of near "zero" in the elution chamber. The sampling port is made of two concentric tubes; an inner capillary(19) inside an outer tube(21). Auxiliary fluid enters from the auxiliary fluid supply line(15), and passes downwards into the outer tube(21), to reach a porous filter(22). Ions of the analytes enter the filter(22) from the electrophoresis gel below(not shown), and are surrounded by the auxiliary fluid which washes the filter. The analytes are passed along with the auxiliary fluid up into the inner capillary(19), to flow through the transfer line(23) to the mass spectrometer. Since the opening of the inner capillary(19) touches the filter(22), the dead volume of the elution chamber is reduced to near-zero. The filter can optionally be filled with gel. The entire tubing system is supported at its junctions by metal braces(24), and secured in place by O-rings(25) and fasteners(26) present at the tubing junctions.

In one preferred embodiment of the apparatus, the stream of auxiliary fluid is pumped in a periodic on/off way. When the flow of the auxiliary liquid is stopped, charged molecules still move by electrophoresis into the elution chamber above the sampling port and accumulate there. During the pumping phase the liquid is moved toward the MS. Such flow configuration increases the maximal concentration of analyte that reaches the MS. Only minute amounts of fluid, measured in microliters, are pumped into the capillary during each pumping spurt, to prevent dilution of the analytes with auxiliary fluid.

A second auxiliary fluid can be introduced to the tubing before the tubing connects to the mass spectrometer, from a second supply line for auxiliary liquid. This second auxiliary fluid can be a solvent that promotes ionization of analytes, and thus increases the sensitivity of the mass spectroscopy measurements.

Example 1

An aqueous solution containing amino acids and a dye compound, was prepared to be separated by electrophoresis in an agarose gel. This analyte solution contained 0.1M aspartic acid, 0.1M glutamic acid, 0.1 phthalic acid and 0.1M fluorescein, adjusted to pH 9.2 using NH OH.

An agarose gel was prepared by addition of 0.8gr agarose to 10ml of (0.1M ammonium acetate- NH OH, pH 9.2), and heating to 80°C. The hot solution was poured into a 7 x 3.5 x 0.4cm electrophoresis cell, a gel comb was inserted, and the solution was allowed to cool and harden to a gel.

5μl of the analyte solution were pipetted into a well in the agarose gel, and electrophoresis was performed in an electrophoretic cell for 15 minutes at 100V (using a CONSORT-862 power supply).

A sampling port was created by filling a capillary of inactivated guard column, having a diameter of 0.53 mm, and a length of 2.8cm, with polyacrylamide gel according to the following procedure:

The polyacrylamide gel was prepared using several solutions of the following compositions:

"A" - 60 g acrylamide + 1.6 bisacrylamid dissolved in 200 ml double distilled water.

"B" - 8 g potassium peroxide sulfate in 100 ml of double distilled water

"C"- TEMED ( N,N,N',N'-tetramethylethylenediamine) - catalyst for acrylamide polymerization

"D"- 0.1 M aqueous ammonium acetate.

The capillary was filled with the polyacrylamide gel by mixing as follows: 5 microliter of "A", 80 microliter of "B", 2 ml of "D" were mixed and deaerated by flushing with nitrogen. 10 microliter "C" was added to the mixture, stirred for few seconds and the solution was drawn into syringe. The sampling capillary was attached to the syringe and filled with solution. Gelling of solution inside capillary was accomplished in 15 minutes. The gel-filled capillary was clamped into a stainless steel capillary coupler, and connected at one end to a tubing system.

Referring to Figure 5, the apparatus used in Example 1 is depicted. The analyte mixture underwent electrophoresis in the agarose gel, while the gel(2) was supported by a metal plate(13). The metal plate is connected at one end to a Consort-862 power source(27); upon activation of the power source, the metal plate will act as the cathode. The gel-filled capillary(l) which acts as a sampling port, is positioned vertically above the agarose gel(2), in close proximity to the wells. At the start of the experiment, the capillary was inserted to a depth of 1mm inside the gel. The capillary is held at its upper end by a T-shaped stainless steel coupler (28). A stainless steel tube(29) enters the coupler from another branch in the coupler's T-junction, perpendicular to the capillary. An opening in this stainless steel tube(29) mates with the upper end of the capillary(l), so that materials passing through the capillary can exit from the upper end of the capillary and enter the lumen of the stainless steel tube(29). The stainless steel tube is com ected at one end through tubing(30) to a syringe pump(31) containing 0.05 M NH CH3COOH-NH4OH, pH 9.2, which acts as auxiliary fluid. The stainless steel tube(29) is connected at its other end to a transfer line(32) which leads towards a Finnigan™ electrospray ionization mass spectrometer(6). A second coupler(33) clamps and holds together the ends of the transfer line(32) and the stainless steel tube(29). The stainless steel tube(29) is connected in addition to the Consort-862 power source (27), and upon activation of the power source, the stainless steel tube will act as the anode. A syringe pump(12) containing methanol is attached to the tubing(32) that leads to the mass spectrometer(6), via another T-shaped coupler(lθ).

The two syringe pumps(31), (12), were activated, to release their respective fluids into the tubing system at a rate of 2 μl per min. and 4 μl per min., respectively. The power source(27) was activated and a voltage of 175V was applied between the electrodes. The negatively charged ions of the three amino acids and of fluorescein, were attracted from within the agarose gel(2) towards the anode(29), and thus migrated in response to the electrical current into the receiving capillary(l) and through it. The ions exited the upper end of the capillary(l) and traveled via the tubing(32) towards the mass spectrometer(6). The auxiliary fluid pumped from its syringe pump(31) accelerated passage of the ions towards the mass spectrometer, and the methanol pumped from its syringe pump(12) surrounded the ions with an environment appropriate to mass spectrometry analysis.

The capillary(l) was repeatedly repositioned relative to the agarose gel, by advancing the capillary forward along the gel, to scan the gel at a rate of 0.5mm per minute. Thus the electrical voltage was constantly applied to a new position in the agarose gel, and ions were released from different bands within the gel, which represent different materials that underwent separation in the gel.

Mass spectral data was recorded continuously during the experiment. Referring to Figure 6, the mass spectral data is shown for of each of the four acids (three amino acids and fluorescein) that were analyzed in Example 1 using the apparatus described. Each chromatogram plots the relative abundance of one of the four acids, in relation to the distance in millimeters on the agarose gel.

Figure 6A is the chromatogram of aspartic acid, Figure 6B is the chromatogram of glutamic acid, Figure 6C is the chromatogram of phthalic acid, and Figure 6D is the chromatogram of fluorescein. Each chromatogram is dependant upon the specific mass to charge ratio (rn/z) which correlates to that acid.

Figure 6 illustrates the dependence of relative abundance of protonated molecules of each of the three amino acids and fluorescein on position of the sampling capillary relative to the starting point of electrophoresis on the gel plate. The four chromatograms corresponding to the m/z of the four acids show that the sampling instrument coupled with the ESI/MS can readily identify the four different negatively charged bands in the gel.

Since prior to the mass spectroscopy analysis, these four acids were partially separated by electrophoresis on an agarose gel into "fuzzy", partially- overlapping bands, the relative abundance of the protonated forms of each of the four acids is shown to be dependant upon the position of the receiving capillary in relation to these bands on the agarose gel. Even though the bands overlapped in the gel, and the separation in the gel was not complete, after the acids were made to migrate from the gel, using the apparatus, to the mass spectroscope, and analyzed by ESI-mass spectroscopy, the four acids can readily be identified. This can be seen for instance, when the chromatogram of aspartic acid, in Figure 6A, is compared to that of glutamic acid, in Figure 6B. Both acids were removed from the gel when the capillary was held over the gel at a distance of approxim25-35mm from the edge of the gel, and though the peaks in the respective chromatograms partially overlap, the two amino acids were easily identified by ESI-mass spectroscopy analysis. Example 2

The apparatus used in this example was as described in Example 1, with the following exceptions:

The buffer used to prepare the agarose gel was 0.025M ammonium acetate, pH5.

The receiving capillary was a poraplot™ packed chromatography column (Chrompack), having an inner diameter of 0.32mm, and a length of 1.4cm.

An aqueous solution of Cytochrome C was prepared by dissolving Cytochrome C in 0.025 ammonium acetate, to a final Cytochrome C concentration 0.02g/ml. The pH was adjusted to pH5 using acetic acid.

7μl of this solution were pipetted into a well in the agarose gel, and electrophoresis was performed in the horizontal plane by application of 100V DC voltage for 30 minutes. The Cytochrome C was noted to have migrated 1.5cm from the well, and was seen as a colored spot in the gel.

The electrophoresis cell with the gel inside was placed below the displaceable sampling port. The receiving capillary was positioned with one opening above the center of this colored spot, and electrophoresis was performed in the vertical plane at 600V for 15 minutes.

Referring to Figure 7, the relative abundance of three representative ions of Cytochrome C are plotted in relation to the time of analysis, on three chromatograms. Each chromatogram is dependant upon the specific mass to charge ratio (m/z) which correlates to that ion. Figure 7A represents an ion characterized by an m/z ratio of 1377, Figure 7B represents an ion characterized by an m/z ratio of 1549, and Figure 7C represents an ion characterized by an m/z ratio of 2065. Since the molecular weight of Cytochrome C is 12384, these ions correspond to "z" equals 10, 8, and 6, respectively. Figure 7D plots the mass spectrum of Cytochrome C.

This example demonstrates that a positively charged protein can be made to migrate from an electrophoresis gel to a mass spectrometer using the method and apparatus according to the present invention. The protein can then be successfully identified in the mass spectrometer.

Claims

1. A method for coupling of planar chromatography and mass spectroscopy, comprising: a) placing a matrix that contains analytes that have been separated by planar chromatography in a gel, between at least two electrodes; b) activating an electrical current in said electrodes, wherein activation of the current induces movement of said analytes out of the matrix, towards one of said electrodes; c) collecting the analytes from the external surface of said matrix to a sampling port; d) transferring the analytes to a mass spectrometer.
2. A method according to claim 1, wherein the matrix is an electrophoresis gel.
3. A method according to claim 1, wherein the matrix is essentially comprised of polyacrylamide.
4. A method according to claim 1, wherein the analytes are selected from the group consisting of polypeptides, proteins, nucleic acids, amino acids, inorganic polymers, organic polymers or derivatives thereof.
5. A method according to claim 1, wherein said electrodes are located above and below said matrix.
6. A method according to claim 1, wherein collection of the analytes from the surface of the matrix is performed in an automated process.
7. A method according to claim 1, wherein the transfer of analytes to a mass spectrometer is performed in an automated process.
8. A method according to claim 1, wherein the electrical current activated is sufficient to produce an electrical field between said electrodes of 1-1000 V/cm.
9. A method according to claim 1, wherein the matrix is of a type selected from the group consisting of an agarose gel, or a polyacrylamide gel.
10. A method according to claim 1, wherein the analytes separated by planar chromatography in the matrix, have underwent a procedure selected from the group consisting of isoelectric focusing, two-dimensional electrophoresis, or one-dimensional electrophoresis.
11. An apparatus for coupling of planar chromatography and mass spectroscopy, according to the method defined in claim 1, comprising: a) a plate for holding a planar chromatography matrix; b) at least two electrodes; c) a power source for supplying voltage between said electrodes; d) at least one displaceable sampling port, wherein said sampling port is oriented perpendicular to said plate; e) means for producing movement of the sampling port relative to said plate; f) a mass spectrometer and an accompanying computer processor; g) a tubing system attached at one end to said sampling port, wherein said tubing system allows passage of analytes from said sampling port to said mass spectrometer.
12. An apparatus according to claim 11, wherein the sampling port is a capillary.
13. An apparatus according to claim 11, wherein the sampling port is a porous filter.
14. An apparatus according to claim 11, wherein one of the electrodes is displaceably located within the planar chromatography matrix.
15. An apparatus according to claim 11, wherein at least one of the electrodes is displaceably located within the sampling port.
16. An apparatus according to claim 11, wherein one of said at least two electrodes is displaceably located adjacent to the sampling port.
17. An apparatus according to claim 11, wherein the means for producing movement of the sampling port are comprised of computer-controlled robotic arms.
18. An apparatus according to claim 11, additionally comprising at least one pump and at least one reservoir of auxiliary fluid; wherein said pump is attached to said at least one reservoir and to said tubing system, and said pump is capable of delivering auxiliary fluid from said reservoir into said tubing system.
19. An apparatus according to claim 11, wherein the displaceable sampling port contains wet gel.
20. An apparatus according to claim 11, wherein the displaceable sampling port contains a porous filter in the tip of said sampling port proximal to the matrix plate.
21. An apparatus according to claim 11, wherein the displaceable sampling port contains a gel plug in the tip of said sampling port proximal to the matrix plate, wherein said gel plug is capable of preventing flow of analytes from inside the sampling port to said plate.
22. An apparatus according to claim 11, wherein the mass spectrometer is selected from the group consisting of an inductive coupled plasma mass spectrometer, an electrospray ionization mass spectrometer, or an atmospheric pressure chemical ionization mass spectrometer.
23.An apparatus according to claim 11, wherein the gel plate and the sampling port are enclosed within a pressure chamber.
24. An apparatus according to claim 11, further comprising a dialysis membrane within the tubing system.
25. An apparatus according to claim 11, further comprising a cation exchange membrane within the tubing system.
26. An apparatus according to claim 11, further comprising an anion exchange membrane within the tubing system.
PCT/IL2001/000521 2000-06-07 2001-06-06 Method and apparatus for direct coupling of planar chromatography with mass spectrometry WO2001094931A2 (en)

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