JP4182789B2 - Method for analyzing low molecular weight protein contained in a test solution derived from a biological sample by electrophoresis and use thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an analysis method for electrophoresis of a low molecular weight protein contained in a test solution derived from a biological sample, and use thereof.
[0002]
[Prior art]
In the fields of medicine and bioscience, for example, analysis of proteins contained in trace amounts in biological samples such as blood is important for testing and screening based on proteins that are disease markers and for exploratory research for proteins that are novel biomarkers. is there. These useful proteins include low molecular weight proteins that are contained only in trace amounts in the biological sample.
For example, when the biological sample is blood, the biological sample contains many proteins. Specifically, albumin, α ₂ High molecular weight proteins such as macroglobulin and transferrin account for more than 80% of the total protein. Therefore, if the biological sample is electrophoretically separated without any pretreatment and the separated protein is detected, the high molecular weight protein as described above is detected excessively, and the low molecular weight protein contained in a trace amount is detected. (For example, refer nonpatent literature 1).
As a pretreatment method as described above, for example, there is known a method of removing albumin contained most in the biological sample by utilizing that albumin specifically adsorbs to Cibacron Blue dye ( For example, refer nonpatent literature 2).
[0003]
[Non-Patent Document 1]
Georgiou et al., Proteomics, 1, 1503-1506 (2001)
[0004]
[Non-Patent Document 2]
Metcalf et al., Biochem. J., 199, 465-472 (1981)
[0005]
[Problems to be solved by the invention]
However, since the affinity between albumin and cibacron blue dye differs depending on the animal species, there are few effects depending on the animal species. Furthermore, the method using cibacron blue dye mainly removes albumin, α ₂ High molecular weight proteins such as macroglobulin and transferrin could not be removed. As a result, even when the pretreated biological sample is electrophoretically separated and the separated protein is detected, the high molecular weight protein is still detected excessively, and the low molecular weight protein contained in a trace amount can be detected. could not.
[0006]
[Means for Solving the Problems]
As a result of various studies on the analysis method of low molecular weight proteins contained in a test solution derived from a biological sample by electrophoresis, the present inventors have obtained a secondary sample from which a high molecular weight protein has been removed from a primary sample by specific pretreatment. The secondary sample is electrophoretically separated, and the separated protein having a molecular weight of 5,000 to 60,000 daltons is detected, so that a low molecular weight protein that is contained in a trace amount in the biological sample is detected. The inventors have found that an analysis method by electrophoresis is possible, and have reached the present invention.
[0007]
That is, the present invention
1. A protein having a molecular weight of 5,000 to 60,000 daltons (hereinafter referred to as the present low molecular weight) contained in a test liquid derived from a biological sample (hereinafter also referred to as the present biological sample) (hereinafter also referred to as the present test liquid). (Sometimes referred to as protein)) by an electrophoresis method,
(1) Particles having a small space in contact with the outside while being continuous with the inside of the particle, and the other outer surface of the particle other than the outer surface in the small space has higher hydrophilicity than the outer surface in the small space ( Hereinafter, the primary particles may be referred to as the present particles), the first step of contacting the test liquid as the primary sample,
(2) a second step in which a polar solvent is brought into contact with the particles obtained in the first step to form a secondary sample; and
(3) Third step of electrophoretic separation of the secondary sample formed in the second step and detecting the separated protein
(Hereinafter, also referred to as the present analysis method);
2. The method according to item 1 above, wherein the method of electrophoretic separation of the secondary sample in the third step is one-dimensional electrophoresis or two-dimensional electrophoresis;
3. 3. The analysis method according to item 1 or 2, wherein the method for electrophoretic separation of the secondary sample in the third step is polyacrylamide gel electrophoresis;
4). A method for obtaining a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test solution derived from a biological sample, the method comprising the step of recovering the protein detected by the method according to item 1 above ( Hereinafter, it may be described as a method for obtaining the present invention.);
5. A method for identifying a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test liquid derived from a biological sample,
(1) a step of recovering the protein detected by the method according to item 1 or a peptide obtained by fragmenting the protein by peptide degradation;
(2) A step of mass spectrometry of the protein or fragmented peptide recovered in the previous step
An identification method characterized by having (hereinafter also referred to as the present invention identification method);
Etc. are provided.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The analysis method of the present invention is an analysis method by electrophoresis of a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test solution derived from a biological sample, and (1) a small space that is in contact with the outside while being continuously connected inside the particle. And a first step of bringing the test liquid that is the primary sample into contact with particles having a higher hydrophilicity than the outer surface in the small space, the other outer surface of the particles other than the outer surface in the small space, (2) a second step in which a polar solvent is brought into contact with the particles obtained in the first step to form a secondary sample, and (3) the secondary sample formed in the second step is subjected to electrophoretic separation, Third step of detecting the separated protein
It is characterized by having.
[0009]
Examples of the “biological sample” (that is, the present biological sample) in the analysis method of the present invention include, for example, various biological cells or tissues containing the same (here, tissues include body fluids such as blood, plasma, serum, and lymph, lymph And any biological sample such as body secretions (urine, milk, etc.). Representative biological samples include body fluids such as blood, plasma, serum and lymph.
In the analysis method of the present invention, the “biological sample-derived test solution” (that is, the present test solution) may be used as it is or a diluted solution thereof as the test solution if the above-described biological sample is in a liquid state. If a biological sample (liver tissue, muscle tissue, brain tissue, heart tissue, etc.) is in a non-liquid state, for example, a biological sample prepared in a liquid state (generally by homogenization, filtration and / or centrifugation, etc.) Is sometimes referred to as “tissue extract”) or a diluted solution thereof may be used as a test solution.
The diluted solution of the biological sample may contain a polar solvent described later, and may be an acidic, basic, or neutral aqueous solution or aqueous buffer containing the polar solvent.
[0010]
The “particle” (that is, the present particle) used in the first step of the analysis method of the present invention is a particle having a small space in contact with the outside while being continuous with the outside, and particles other than the outer surface in the small space. The other outer surface is a particle having higher hydrophilicity than the outer surface in the small space. Here, the particles having “a small space in contact with the outside while being continuous inside” are, for example, generally referred to as porous particles, and the particle diameter thereof is about 0.5. Spherical (i.e., in the form of beads) in the range of ~ 500 [mu] m (preferably about 20-200 [mu] m) is preferred. The specific surface area of the particles is, for example, about 200 to 800 m. ² / G is preferred.
In addition, “the other outer surface of the particle other than the outer surface in the small space has higher hydrophilicity than the outer surface in the smaller space” means, for example, a particle other than the outer surface in the smaller space existing inside the particle. The outermost surface (that is, the outermost surface in contact with the outside of the particle) has a hydrophilic group mainly, and the outer surface in a small space existing inside the particle (that is, the outer surface existing inside the particle) Particles mainly having a hydrophobic group or an ion-exchange group, and having a structure in which the outermost surface in contact with the outside of the particle has a hydrophilic group such as a diol group are preferable. The selection of the particles is appropriately determined by preliminary experiments. Generally, for the low molecular weight protein having a relatively low polarity, particles having a hydrophobic group on the outer surface existing inside the particles are used. For this low molecular weight protein having a relatively high polarity, particles having an ion exchange group on the outer surface present inside the particles are preferably used.
[0011]
The particles are produced by a method similar to the method for producing a water-wettable polymer formed by copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers. be able to.
Here, the “water-wettable polymer” represents a polymer that is partially or wholly solvated by water. "Monomer" refers to a prepolymerized molecule containing one or more polymerizable functional groups and a repeating structural unit of the polymer. A “polymer” may contain two or more different monomers, in this case also called a “copolymer”. “Mole%” of a monomer contained in the copolymer means the mole fraction (expressed in%) of the monomer with respect to the total number of moles of the various (two or more) monomers constituting the copolymer. Yes.
The hydrophobic monomer may include a hydrophobic moiety. Preferred hydrophobic moieties include, but are not limited to, phenyl groups, phenylene groups, or alkyl groups having 2 to 18 carbon atoms. More specifically, for example, divinylbenzene, styrene and the like are more preferable hydrophobic monomers.
The hydrophilic monomer may include a hydrophilic portion. Preferable hydrophilic moieties include saturated, unsaturated or aromatic heterocyclic groups (specifically, for example, pyrrolidonyl group, pyridyl group, etc.). Another preferred hydrophilic moiety is an ether group. More specifically, for example, N-vinyl pyrrolidone, 2-vinyl pyridine, 3-vinyl pyridine, 4-vinyl pyridine, ethylene oxide and the like are more preferable hydrophilic monomers.
For example, when the present particles are a porous polymer made of a poly (divinylbenzene-co-N-vinylpyrrolidone) copolymer, a normal radical polymerization method as described in, for example, US Pat. No. 4,382,124 is used. Then, it may be produced by copolymerizing divinylbenzene and N-vinylpyrrolidone.
The composition of the resulting copolymer is determined by the initial stoichiometry of the above two monomers, but the starting material ratio may not be substantially the same due to reactivity differences between the monomers. is there. When the present particles are a porous polymer composed of a poly (divinylbenzene-co-N-vinylpyrrolidone) copolymer, for example, about 5 to 30 mol% (preferably about 10 to 25 mol%, more preferably 15 to 20 mol%). A preferred recovery rate of the present low molecular weight protein can be obtained in a composition containing about N) -vinylpyrrolidone.
[0012]
In the first step of the analysis method of the present invention, in order to bring the present test solution, which is a primary sample, into contact with the present particles, any method that enables intimate contact between the present particles and the present test solution (for example, a batch process or a chromatographic process). The test liquid may be brought into contact with the particles in a photographic process). For example, the test solution may be forcibly passed through a column, disk, or plug filled with the particles, or the test solution may be stirred with the particles, for example, in a batch agitation reactor. The test solution may be further added to a polymer-containing well of a microtiter plate. The particles may take the form of beads or pellets, for example. The test solution is contacted with the particles for a time sufficient for the low molecular weight protein to be adsorbed onto the particles. The time is usually the time required for the low molecular weight protein to equilibrate between the outer surface of the particle and the test solution. The adsorption or distribution of the low molecular weight protein on the particles can be partial or complete.
The particles are treated with a water-miscible organic solvent prior to contact with the test solution, then with water or an aqueous buffer, and then the test solution is contacted with the particles. Alternatively, the test liquid may be brought into contact with the particles in a state where the particles are not wet (ie, the particles are dried) before the contact with the test liquid.
[0013]
When the test solution is supplied to a column filled with the present particles so that the test solution is brought into contact with the particles, the particles are filled into a container having an open end to form a solid-phase extraction cartridge. . The container may be, for example, a cylindrical container open at both ends so that the test solution enters the container from one end, contacts the particles in the container, and exits the container from the other end. It may be a column. The particles can be filled into a container. The solid-phase extraction cartridge is an insoluble solid in order to hold the particles in the cartridge and to allow the test solution to enter and exit from the cartridge when the test solution flows into the cartridge. In order to remove the substance from the test solution, a porous holding means (for example, a filter element or a frit) may be further included at one or both ends of the cartridge adjacent to the particle. Such a filter can be made, for example, from molten glass or a porous polymer (eg, porous high density polyethylene).
The solid phase extraction cartridge may be a single use cartridge that is used in the processing of a single sample and then discarded, or it may be a cartridge that can be used to process multiple samples.
The amount of the particles in the container is limited by the volume of the container and is preferably in the range of about 0.001 g to about 50 g, more preferably about 0.025 g to about 1 g. The amount of the present particles may be appropriately determined according to the amount of the present low molecular weight protein to be adsorbed, the effective surface area of the present particles, and the strength of the interaction between the present low molecular weight protein and the present particles. .
[0014]
In the case where the test solution is supplied to a column packed with the present particles to bring the test solution into contact with the present particles, the solid phase extraction formed by filling the particles into a container whose end is opened as the column. When the cartridge is used, the test liquid is added to the upper part of the solid-phase extraction cartridge, and the test liquid to be adsorbed is caused to flow through the cartridge in a natural flow state and contact the main particles. The test liquid can flow through the cartridge according to gravity. Increasing the flow rate can be achieved by creating a pressure differential across the cartridge. Such a pressure difference is obtained by attaching a pressure source to the lower end of the cartridge and applying a positive pressure to the upper end of the cartridge (for example, applying pressurized gas (for example, air or nitrogen) to the upper portion of the cartridge). Or by compressing the air in the cartridge above the particles with a piston or plunger. The flow rate of the test liquid passing through the cartridge can be changed by adjusting the pressure difference between both ends of the cartridge.
[0015]
The flow rate and flow time of this test solution vary depending on the type of the low molecular weight protein, the type of the column, etc., and the optimum conditions are appropriately determined by preliminary experiments, but generally about 0.5 to 2 ml / By passing the liquid for about 0.5 to 10 minutes, the low molecular weight protein contained in the test liquid can be adsorbed in the particles. An appropriate solution flow rate (indicated by the linear velocity of the solution) can be, for example, about 14 mm / second at the maximum, and preferably about 0.7 to 3.5 mm / second. In this way, only the low molecular weight protein contained in the test solution is adsorbed in the particles while removing the high molecular weight protein contained in the test solution.
[0016]
As such a column, for example, Oasis HLB (trade name) (manufactured by Waters), L-column L-1180 (trade name) (manufactured by Chemicals Inspection Association), Develosil PT C8-5 (trade name) (Nomura Chemical) ), Shim-pack SPCAE1 (trade name) (manufactured by Shimadzu Corporation), TSK precolumn PW (trade name) (manufactured by Tosoh Corporation), and the like.
[0017]
In the second step of the analysis method of the present invention, in order to form a secondary sample by bringing the particles obtained in the first step into contact with a polar solvent, any particle that allows intimate contact between the particles and the polar solvent can be used. The particles may be contacted with a polar solvent by a method (eg, a batch process or a chromatographic process).
[0018]
For example, when the test liquid that is the primary sample is supplied to the column packed with the main particles in order to bring the test liquid that is the primary sample into contact with the particles in the first step, a polar solvent is passed through the column. What is necessary is just to liquid. When a solid phase extraction cartridge formed by filling the particles in a container with an open end is used as the column, a polar solvent is added to the top of the solid phase extraction cartridge, In this state, the particles are brought into contact with a polar solvent. Polar solvent can flow through the cartridge according to gravity. Increasing the flow rate can be achieved by creating a pressure differential across the cartridge. Such a pressure difference is obtained by attaching a pressure source to the lower end of the cartridge and applying a positive pressure to the upper end of the cartridge (for example, applying pressurized gas (for example, air or nitrogen) to the upper portion of the cartridge). Or by compressing the air in the cartridge above the particles with a piston or plunger. The flow rate of the polar solvent through the cartridge can be changed by adjusting the pressure difference across the cartridge.
[0019]
The flow rate and flow time of the polar solvent vary depending on the type of the primary sample, the type of the column, etc., and the optimum conditions are appropriately determined by preliminary experiments, but generally about 0.1 to 2 ml / min, preferably Is passed through the column at a rate of about 0.1 ml / min to 1 ml / min, more preferably about 0.2 to 0.5 ml / min for about 0.5 to 15 minutes, preferably about 5 to 10 minutes. The passed polar solvent can be recovered to form a secondary sample. An appropriate solution flow rate (indicated by the linear velocity of the solution) can be, for example, about 14 mm / second at the maximum, and preferably about 0.7 to 3.5 mm / second. In this manner, the particles are washed with a polar solvent so that the low molecular weight protein adsorbed in the particles is desorbed or eluted from the particles, thereby forming a secondary sample containing the low molecular weight protein. The
[0020]
The polar solvent used in the second step in the analysis method of the present invention is not particularly limited as long as it is a polar solvent suitable for the combination with the present particles used in the first step, but a polar solvent not containing inorganic salts is preferably used. It is done.
Examples of the polar solvent include a polar water-miscible organic solvent or a water / organic solvent mixture, or a liquid containing water or an organic acid in water. Examples of the polar water-miscible organic solvent include alcohols such as methanol, ethanol and isopropanol, acetonitrile, acetone, tetrahydrofuran and the like, and examples of the organic solvent used in the water / organic solvent mixture include the above-mentioned Nonpolar or moderately polar water-immiscible solvents (for example, dichloromethane, diethyl ether, chloroform, or ethyl acetate) are also included. Further, a mixed solution of these solvents may be used. In addition, by using a polar water-miscible organic solvent, it is described in McDonald and Bouvier, supra, (1995); Snyder and Kirkland, Introduction to Modern Liquid Chromatography, New York: J. Wiley and Sons (1974), etc. By using this information, it is possible to determine an appropriate solvent without undue experimentation. For example, acetonitrile or methanol can be particularly preferred. Generally, the content of the polar water-miscible organic solvent is about 50 to 100% by weight, preferably 50 to 80% by weight, more preferably about 50 to 60% by weight.
[0021]
Examples of polar solvents include polar water-miscible organic solvents or water / organic solvent mixtures, and examples of organic acids contained in water include butyric acid, propionic acid, acetic acid, formic acid, and trifluoroacetic acid. Can do. Preferred are acetic acid, formic acid, trifluoroacetic acid and the like. The amount of the organic acid contained can be about 0.05 to 2% by weight. Preferably about 0.1 to 1% by weight, more preferably 0.1 to 0.2% by weight.
[0022]
The electrophoresis used in the third step in the analysis method of the present invention may be any electrophoresis method that can separate proteins, and is not particularly limited. In addition, when removing the organic solvent and organic acid which are contained in a secondary sample as needed, it can make easily by drying, for example. Then, before being subjected to electrophoresis, the dried secondary sample may be redissolved in an appropriate solvent.
[0023]
For example, after the secondary sample dried in a solution containing SDS is dissolved again, this can be subjected to one-dimensional electrophoresis using SDS polyacrylamide gel to separate the low molecular weight protein. In addition, after the secondary sample is dissolved again in a solution containing urea or thiourea, it is subjected to isoelectric focusing in the first dimension and then subjected to SDS polyacrylamide gel electrophoresis in the second dimension. The low molecular weight protein can also be separated by electrophoresis.
[0024]
In order to detect this low molecular weight protein separated in the third step in the analysis method of the present invention, any detection method capable of detecting the protein may be used, and there is no particular limitation. For example, the low molecular weight protein can be stained and detected with the dye Coomassie Brilliant Blue R250.
Detection of the stained low molecular weight protein may be performed by visual observation, densitometer, image analysis system or the like from the electrophoretic image. Examples of the image analysis system include PDQuest from Bio-Rad and ImageMaster 2D from Pharmacia.
When analyzing the electrophoretic image, for example, the low molecular weight protein can be detected based on the difference between the electrophoretic image of the test solution and the electrophoretic image of the control.
[0025]
Thus, this low molecular weight protein can also be acquired from this test solution by collect | recovering this low molecular weight protein detected by this invention analysis method. For example, in the electrophoresis used in the third step in the analysis method of the present invention, when a polyacrylamide gel is used, the low molecular weight protein can be easily extracted and recovered from the gel. The extraction method is not particularly limited. For example, conventional methods such as a method of fragmenting and extracting the low molecular weight protein into peptides in the gel using trypsin, a method of electrophoretic transfer to the membrane and extraction, etc. can give.
[0026]
Furthermore, the low molecular weight protein contained in the test solution is identified by recovering the low molecular weight protein detected by the analysis method of the present invention or a peptide obtained by fragmenting the protein by peptide degradation. You can also. The method for identifying the low molecular weight protein is not particularly limited. For example, the fragmented peptide is mass-reduced by an ion trap mass spectrometer or a quadrupole-time-of-flight mass spectrometer using an ESI ionization method or the like. Analysis or mass analysis may be performed with a time-of-flight mass spectrometer using a MALDI ionization method or the like. Alternatively, after electrophoretic transfer to a membrane and extraction, the protein may be immunoassayed using an antibody specific for the low molecular weight protein.
[0027]
【Example】
EXAMPLES Hereinafter, although an Example etc. demonstrate this invention further in detail, this invention is not limited at all by those Examples.
[0028]
Example 1 (Preparation of this test solution)
Mouse serum (manufactured by Sigma-Aldrich Japan) was used as the biological sample. Human growth hormone (manufactured by Sigma-Aldrich Japan), myoglobin (manufactured by Sigma-Aldrich Japan), ovalbumin (manufactured by Sigma-Aldrich Japan), 10 μg each were added to 100 μl of mouse serum, and 0.1% trifluoroacetic acid (TFA) (TFA) ( This test solution was prepared by adding an aqueous solution (Applied Biosystems Japan) to a total volume of 1 ml.
[0029]
Example 2 (Preparation of the present particles: Production of poly (divinylbenzene-co-N-vinylpyrrolidone) copolymer)
A solution obtained by dissolving 50 g of hydroxypropylmethylcellulose (Dow Chemical) in 1000 ml of water is added to a 3000 ml flask. 175 g of divinylbenzene (DVB HP-80, manufactured by Dow Chemical), 102 g of N-vinyl-2-pyrrolidone (available from International Specialty Products) and 1.85 g of azobisisobutyronitrile (Vazo64, manufactured by DuPont Chemical) ) In 242 g of toluene is added to the above mixture. The resulting biphasic mixture is thoroughly stirred at room temperature for 30 minutes to form the desired micron-sized oil droplets. The resulting suspension is then heated to 70 ° C. with moderate stirring and held at that temperature for 20 hours. The suspension is then cooled to room temperature, filtered and washed with methanol. The resulting filter cake is dried at 80 ° C. under reduced pressure for 16 hours. The composition of the polymer product is determined by elemental analysis. By varying the starting ratio of divinylbenzene monomer to N-vinylpyrrolidone monomer, a series of poly (divinylbenzene) containing about 13, 14, 16 and 22 mol% N-vinylpyrrolidone according to the method described in this example. -Co-N-vinylpyrrolidone).
By filling 50 mg of each polymer thus produced into a 1 cc Sep-Pak VacR cartridge container (manufactured by Waters Corporation) that requires polyethylene frit at the inlet and outlet of the polymer layer, the solid phase extraction cartridge is Let it form.
[0030]
Example 3 (Preparation of the present particles)
A commercially available Oasis HLB (trade name) (manufactured by Waters) was used as a column filled with 30 mg of the present particles in a 1 ml syringe. The particles were washed by passing an 80% acetonitrile (manufactured by Kanto Chemical) aqueous solution containing 0.1% trifluoroacetic acid through the column. Next, the particles were equilibrated by passing an aqueous solution containing 1 ml of 0.1% trifluoroacetic acid.
[0031]
Example 4 (First step in the analysis method of the present invention)
1 ml of this test solution prepared in Example 1 was passed at room temperature at a flow rate of 1 ml / min through a column filled with syringes with the present particles equilibrated in Example 3.
Next, the particles were washed by passing 1 ml of an aqueous solution containing 0.1% trifluoroacetic acid through the column thus obtained.
[0032]
Example 5 (Second step in the analysis method of the present invention)
1 ml of an 80% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid is passed through a column packed with the main particles washed in Example 4 in a syringe, and the eluate discharged from the column is taken as a secondary sample. As recovered.
[0033]
Example 6 (Third step in the analysis method of the present invention)
The recovered eluate (that is, the secondary sample) was dried with a SpeedVac concentrating dryer (manufactured by Servant) to obtain a dried secondary sample while removing trifluoroacetic acid and acetonitrile.
Subsequently, the secondary sample thus obtained was re-dissolved in 1 ml of water, and the sample for electrophoresis and the test solution prepared in Example 1 were subjected to one-dimensional electrophoresis using SDS polyacrylamide gel. This low molecular weight protein was separated by subjecting to electrophoresis (electrophoresis for 40 minutes at a constant voltage of 200 V in Tris-glycine buffer). As the gel, an SDS polyacrylamide gel (manufactured by Nippon Bio-Rad Laboratories) having a density gradient of 15 to 25% was used. After the electrophoresis was completed, the gel was immersed in Coomassie Brilliant Blue Staining Kit Quick CBB (trade name) (manufactured by Wako Pure Chemical Industries) for 30 minutes and then washed with water.
The results thus obtained are shown in FIG. In the left lane in the figure (lane (1): electrophoretic image for comparison / this test solution prepared in Example 1), only serum albumin (66 kDa), which is a high molecular weight protein, was detected. Molecular weight proteins myoglobin (16 kDa), human growth hormone (23 kDa) and ovalbumin (45 kDa) could not be detected. On the other hand, in the right lane (lane (2): electrophoresis image for the present invention / electrophoresis sample prepared in Examples 1 to 6) in the figure, myoglobin which is the low molecular weight protein, human growth Hormonal and ovalbumin bands were detected.
[0034]
【The invention's effect】
According to the present invention, an analysis method by electrophoresis of a low molecular weight protein that is contained in a trace amount in a biological sample has become possible.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows an electrophoretic image obtained by separating a protein by one-dimensional electrophoresis using an SDS polyacrylamide gel and then staining the gel with a Coomassie Brilliant Blue staining kit. FIG. In the left lane (lane (1): electrophoretic image for comparison / this test solution prepared in Example 1), only serum albumin (66 kDa), which is a high molecular weight protein, was detected. Molecular weight proteins myoglobin (16 kDa), human growth hormone (23 kDa) and ovalbumin (45 kDa) could not be detected. On the other hand, in the right lane (lane (2): electrophoretic image for the present invention / electrophoresis sample prepared in Examples 1 to 6) in the figure, myoglobin which is the low molecular weight protein, human growth Hormonal and ovalbumin bands were detected.

Claims (5)

An analysis method by electrophoresis of a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test solution derived from a biological sample,
(1) The inside of the particle has a small space that is continuously in contact with the outside, and the other outer surface of the particle other than the outer surface in the small space is a particle having higher hydrophilicity than the outer surface in the small space. A first step of contacting the test liquid, which is a primary sample,
(2) a second step in which a polar solvent is brought into contact with the particles obtained in the first step to form a secondary sample, and (3) the secondary sample formed in the second step is subjected to electrophoretic separation, An analysis method comprising a third step of detecting the separated protein. 2. The analysis method according to claim 1, wherein the method of electrophoretic separation of the secondary sample in the third step is a one-dimensional electrophoresis method or a two-dimensional electrophoresis method. 3. The analysis method according to claim 1, wherein the method of electrophoretic separation of the secondary sample in the third step is polyacrylamide gel electrophoresis. A method for obtaining a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test solution derived from a biological sample, comprising the step of recovering the protein detected by the method according to claim 1. . A method for identifying a protein having a molecular weight of 5,000 to 60,000 daltons contained in a test liquid derived from a biological sample,
(1) a step of recovering the protein detected by the method of claim 1 or a peptide in which the protein is fragmented by peptide degradation; and (2) the protein or fragmented peptide recovered in the previous step. A method for mass spectrometry.

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