JP4847848B2 - Detection method, detection kit and detection apparatus for molecules bound to carrier protein - Google Patents

Description

  The present invention relates to a detection method, a detection kit and a detection apparatus for molecules bound to a carrier protein.

  At present, measurement of proteins (biomarkers) whose expression changes with disease is widely performed as an effective means of clinical examination. However, many of them are not sufficient in sensitivity and accuracy, and the discovery of more effective new biomarkers is desired. With the recent increase in sensitivity of mass spectrometry technology, research for searching for new biomarkers from proteins present in minute amounts in serum has been conducted all over the world.

  However, with conventional methods, trace proteins may not be detected due to proteins (albumin, immunoglobulin, etc.) present at high concentrations in serum. Therefore, conventionally, a method of previously removing a protein present at a high concentration for the purpose of identifying a trace amount of protein has been proposed. Kits for this purpose (Agilent, Multiple Affinity Removal System, GE Healthcare, Albumin and IgG Removal Kit, etc.) are already on the market and are effective tools for analyzing trace protein expression.

  However, since albumin and immunoglobulin are also carrier proteins responsible for protein transport and stabilization, many trace proteins, mainly low molecular weight proteins, are bound to these proteins. Therefore, when an analysis tool such as that described above is used, it is impossible to detect a trace amount of protein that is bound to albumin or immunoglobulin.

  Further, not only albumin and immunoglobulin, but a compound that interacts with a predetermined protein cannot detect the interacting compound even if the protein can be detected. Thus, there is a problem that there is no analysis technique that can detect a compound that interacts with a carrier protein with high accuracy.

  Patent Document 1 discloses a method for purifying a protein complex that interacts with a target protein using microfluidics having a two-stage trap. However, the method disclosed in Patent Document 1 is not disclosed in a technique for isolating a component on an interacting side after purifying a protein complex, and does not solve the above-described problem. Patent Document 2 discloses that after a test substance is bound in a state where the target substance is immobilized on the support, the test substance is removed from the support, and then the target substance is fragmented on the support. And performing mass spectrometry on the same support is disclosed. However, the method disclosed in Patent Document 2 cannot detect a test substance contained in a state of interacting with a target substance contained in a sample, and cannot solve the above-described problem.

WO 2004/029079 JP 2006-98265

  Then, in view of the above-mentioned situation, the present invention has an object to provide a technique that can detect a substance that interacts with a protein present in a sample at a high concentration with high accuracy.

The present invention that has achieved the above-described object includes the following.
The method according to the present invention decomposes a carrier protein separated from a sample, and then selectively removes peptide fragments derived from the carrier protein. Thereby, the method according to the present invention can detect molecules interacting with the carrier protein. That is, the method according to the present invention separates the carrier protein from the sample containing the carrier protein that interacts with the molecule, decomposes the separated carrier protein, and interacts with the peptide fragment derived from the carrier protein and the carrier protein. The molecules that acted are separated and the molecules that interacted with the carrier protein are detected.

  Here, the molecule to be detected in the method according to the present invention is not limited at all, and examples thereof include proteins, sugar chains, nucleic acids, low molecular compounds, organic compounds, drugs, and active substances. The molecule to be detected may be a known compound or an unknown compound. On the other hand, the carrier protein is not particularly limited as long as it is a protein having a function with which the above-described molecules to be detected interact. The carrier protein may be a protein whose function as a carrier protein is known, or may be a protein whose function as a carrier protein is unknown.

  In the method according to the present invention, a method for separating a peptide fragment derived from a carrier protein from the molecule is not particularly limited. For example, a polyclonal antibody against the carrier protein can be used.

  On the other hand, according to the present invention, a kit for separating molecules bound to a carrier protein can also be provided. The kit according to the present invention comprises a first separation means for separating the carrier protein from a sample containing the carrier protein bound with the molecule, a decomposition means capable of decomposing the carrier protein, and the carrier protein obtained by the decomposition means. It comprises a second separation means for separating the peptide fragment produced by decomposition and the molecule bound to the carrier protein. In the apparatus according to the present invention, the second separation means can adjust a sample containing molecules interacting with the carrier protein at a high relative concentration.

  According to the present invention, it is possible to provide a method for detecting a molecule bound to a carrier protein, which can accurately detect a trace component that interacts with a protein present at a high concentration in a sample. . Furthermore, according to the present invention, it is possible to provide a kit for detecting a molecule bound to a carrier protein capable of accurately detecting a trace component that interacts with a protein present in a sample at a high concentration. it can. Furthermore, according to the present invention, there is provided an apparatus for detecting a molecule bound to a carrier protein capable of accurately detecting a trace component that interacts with a protein present at a high concentration in a sample. Can do.

  The method, kit, and apparatus according to the present invention can detect molecules that were difficult to detect by the conventional method, and therefore, a system for evaluating the effects of foods and drugs, pharmacology in the development of new drugs, etc. The present invention can be applied to a system for studying the action.

Hereinafter, the present invention will be described in detail with reference to the drawings.
In the present invention, the molecule to be detected is present in a state of interacting with the carrier protein in the sample. The method according to the present invention detects a molecule interacting with a carrier protein. In the present invention, the carrier protein is not particularly limited, and may be any protein that is known to interact with a specific compound or a protein that is unknown to interact with any compound. . Examples of carrier proteins include albumin, globulin, transferrin, lactoferrin and ferritin.

  In addition, the molecule to be detected in the present invention means a substance that exists in a state of interacting with the carrier protein described above. The type of molecule to be detected is not particularly limited, and examples thereof include substances such as proteins, sugar chains, nucleic acids, low molecular compounds, organic compounds, and drugs including oligopeptides and polypeptides. Further, when albumin or globulin is targeted as the carrier protein described above, examples of the molecule include pigments and drugs such as fatty acids, divalent metal ions, amino acids, hormones, heme, and bilirubin. Furthermore, when transferrin, lactoferrin, or ferritin is used as the carrier protein described above, iron can be used as the molecule. Furthermore, regardless of the type of carrier protein, the molecule to be detected may be a substance whose binding ability and / or function is known, or a substance whose binding ability or function is unknown. .

  In a sample, the state in which a carrier protein and a molecule interact with each other means that the carrier protein and the molecule are integrated by covalent bond, coordination bond, metal bond, ionic bond, hydrogen bond, or bond by intermolecular force. It means the state. That is, the state in which the carrier protein and the molecule interact with each other means a state in which the carrier protein and the molecule exhibit the same behavior regardless of the binding mode.

  The sample may be in any form, for example, a biological sample prepared from blood, serum, plasma, lymph, urine, spinal fluid, saliva, sweat, ascites, amniotic fluid, or a cell or organ extract. Can be used.

  A flowchart of a method for detecting a molecule interacting with a carrier protein to which the present invention is applied is shown as an example in FIG. In this method, as shown in FIG. 1, the step 101 for separating the carrier protein from the sample, the step 102 for decomposing the carrier protein after the separation, the peptide fragment derived from the carrier protein, and the carrier protein were bound. It comprises a step 103 for separating molecules, and a step 104 for detecting molecules bound to the carrier protein. FIG. 2 schematically shows the principle of each treatment in the method for detecting a molecule interacting with a carrier protein to which the present invention is applied.

  In step 101, the target carrier protein 203 is separated from the sample. The means for separating the carrier protein 203 is not particularly limited. For example, a support 201 on which a substance 202 having an affinity for a target carrier protein is immobilized can be used. As a specific method, at least a sample is brought into contact with a surface on which a substance 202 having affinity for a carrier protein in the support 201 is immobilized, and incubated under predetermined conditions (time, temperature, etc.). Thereafter, the sample is removed from the support 201, and components and other contaminants 205 adsorbed nonspecifically on the support are washed away. Then, the carrier protein 203 is separated from the support 201 using a predetermined solution, and the carrier protein 203 is recovered in the solution.

  As the support 201, any solid phase can be used as long as the substance 202 having affinity for the carrier protein can be immobilized. As the support 201, for example, a membrane such as nitrocellulose or PVDF (polyvinylidene fluoride), a glass, a silicon wafer, a resin such as plastic, a metal, or the like is used as a base, and can be fixed to one surface of the base as necessary. What gave appropriate modification can be used. The types of modification include poly-L-lysine modification, which can immobilize the target molecule by physical adsorption, aminosilane modification, and functional groups such as aldehyde group and epoxy group, which can immobilize the target molecule by covalent bond. Avidin modification or Ni-NTA modification that can be immobilized using the modification and affinity with the target carrier protein 203 can be used. Further, a solid phase composed of a thin layer of a hydrophilic porous matrix such as polyacrylamide gel or agarose gel can also be used as the support 201. As the shape of the support 201, a flat substrate such as a slide glass, beads, a column, or the like can be used.

  The substance 202 having affinity for the target carrier protein is appropriately selected according to the type of carrier protein. For example, when albumin is used as the carrier protein 203, a solid phase such as cibacron blue can be used as a substance having affinity for albumin. When globulin is used as the carrier protein 203, protein A, protein G, or the like can be used as a substance having affinity for globulin. In all cases where albumin, globulin, transferrin, lactoferrin, or ferritin is targeted as the carrier protein 203, antibodies, antibody-like substances, aptamers, nucleic acids, nucleic acid-like substances, proteins, peptides, etc. that have affinity for these substances Can be used.

  Means for bringing the support 201 on which the substance 202 having affinity for the carrier protein is immobilized into contact with the sample is appropriately selected according to the shape of the support 201, but is not particularly limited. When the sample is brought into contact with the flat support 201, for example, the sample is supplied into the chamber with the support 201 placed in the chamber. When the sample is brought into contact with the columnar support 201, the sample is injected from the upper opening of the column. Furthermore, when the use is brought into contact with the bead-shaped support 201, the bead-shaped support 201 and the sample are supplied into a desired container and preferably stirred.

  By these treatments, the carrier protein 203 contained in the sample can be captured on the support 201. In addition, following the process described above, the contaminants 205 can be removed from the support 201 by cleaning the support 201 with a cleaning liquid. The washing liquid is not particularly limited, and a washing liquid used in chromatography or the like can be used. Note that the cleaning process using the cleaning liquid can be performed in the same manner as the process of bringing the sample into contact with the support 201 described above. Even after the support 201 is washed with the washing liquid, the target carrier protein 203 can be kept in the state of being captured on the support 201. That is, by this processing, the target carrier protein 203 is captured together with the molecules 204 that interact with the carrier protein.

In addition, as a solution for removing and recovering the carrier protein 203 from the support 201 after the washing treatment, the solution capable of separating the carrier protein 203 captured on the support 201 from the support 201 is used. For example, acids such as 0.1M glycine buffer (pH 2), denaturing agents such as 8M urea, 6M guanidine and 3% SDS, chaotropic ions such as 4.5M MgCl ₂ and 50% ethylene glycol are used. can do. The carrier protein 203 captured on the support 201 by the treatment with the solution is separated and recovered together with the molecules 204 that interact with the carrier protein.

  Next, in step 102, the carrier protein 203 separated from the support 201 is decomposed. Here, the method for degrading the carrier protein 203 is not particularly limited, and examples thereof include a chemical fragmentation method and an enzyme method. Examples of the former include the cyanogen bromide method, and examples of the latter include digestion with trypsin, chymotrypsin, ArgC, AspN, LysC, V8, and the like. It is also effective to use a combination of these.

  By this treatment, the carrier protein 203 becomes a plurality of peptide fragments 206 and releases the interacting molecules 204. Further, when the molecule 204 that interacts with the carrier protein 203 is a protein, it can be similarly converted into a peptide fragment by this treatment.

  Next, in step 103, the peptide fragment 206 derived from the carrier protein 203 generated in step 102 is separated from the molecule 204 that has interacted with the carrier protein 203. The means for separating the peptide fragment 206 derived from the carrier protein 203 and the molecule 204 that has interacted with the carrier protein 203 is not particularly limited. For example, a polyclonal antibody 207 using the carrier protein 203 as an antigen is used. be able to. The polyclonal antibody 207 using the carrier protein 203 as an antigen can be obtained by a conventionally known technique, but commercially available antibodies may be used.

  In order to obtain polyclonal antibody 207 using carrier protein 203 as an antigen, carrier protein 203 purified according to a conventional method is immunized to experimental animals such as mice, rats and rabbits. Experiment animals produce antibodies against carrier protein 203, and then blood is collected, for example, ammonium sulfate precipitation, ion exchange chromatography, affinity column with immobilized protein A or protein G, or affinity with the protein immobilized. The desired polyclonal antibody 207 can be prepared by purification using a column or the like.

  Since the polyclonal antibody 207 having the carrier protein 203 as an antigen thus obtained has various sites in the carrier protein 203 as epitopes, the peptide fragment 206 derived from the carrier protein 203 produced in the above step 102 is specifically identified. Can be adsorbed. Accordingly, in this step 103, the carrier protein contained in the solution is brought into contact with the support 201 on which the polyclonal antibody 207 having the carrier protein 203 as an antigen is immobilized by contacting the solution after the treatment in the step 102. Only the peptide fragment 206 derived from 203 can be specifically adsorbed on the support 201. Thereby, the peptide fragment 206 derived from the carrier protein 203 and the molecule 204 interacting with the carrier protein 203 can be separated from the solution treated in the above step 102.

  Next, in step 104, the molecule 204 that has been bound to the carrier protein 203 is detected. The means 208 for detecting the molecule is not particularly limited, and an appropriate means is selected according to the type of the molecule. Examples of the means 208 for detecting the molecule include an X-ray analyzer, a mass spectrometer, a magnetic resonance apparatus, an electron beam applied analyzer, a surface plasmon resonance apparatus, and various chromatographs. In particular, when identifying an unknown molecule 204 that interacts with the carrier protein 203, it is preferable to use a mass spectrometer.

  The mass spectrometer is not particularly limited, and a mass spectrometer to which any principle is applied can be used. Generally, a mass spectrometer includes a sample introduction unit, an ion source that ionizes peptides and proteins contained in a sample introduced from the sample introduction unit, an analysis unit that separates peptides and proteins ionized by the ion source, and an analysis unit And a data processing unit that generates a mass spectrum from values detected by the detection unit. A liquid chromatography column can be connected to the sample introduction section. Although it does not specifically limit as an ion source, What applied the principles, such as an electron ionization method, a chemical ionization method, a field desorption method, a fast atom collision method, a matrix assisted laser desorption ionization method, and an electrospray ionization method, can be mentioned. it can. The analysis unit is not particularly limited, and examples thereof include a magnetic field deflection type, a quadrupole type, an ion trap type, a time-of-flight type, and a Fourier transform ion cyclotron resonance type, and a tandem type combining these. Also good.

  In this step 104, the molecule 204 that interacts with the carrier protein 203 contained in the sample can be identified by detecting the substance contained in the solution after the treatment in the above step 103. That is, when the molecule 204 that interacts with the carrier protein 203 is unknown, it can be concluded that the molecule 204 detected in this step 104 is a substance that interacts with the target carrier protein 203. For example, when screening a drug or the like that interacts with a target protein for specific disease treatment, symptom improvement, etc., the above-mentioned steps 101 to 104 are carried out using the target protein as the carrier protein 203, thereby causing the disease Candidate compounds for therapeutic and ameliorating agents can be identified.

  In this step 104, the molecules 204 that interact with the carrier protein 203 may be quantified. For example, when albumin is used as the carrier protein, the effect of the drug can be determined by quantifying the albumin-binding drug bound to albumin. Moreover, when the binding substance with respect to the predetermined | prescribed carrier protein 203 is related with a specific disease, the presence or absence and the grade of the said disease can be determined by quantifying the said binding substance.

  By carrying out the above-mentioned steps 101 to 104 on the healthy subject-derived sample and the patient-derived sample, and specifying molecules that change in quantity among the molecules that interact with the carrier protein, a carrier protein-binding disease marker Can be identified.

  In addition, the above-described steps 101 to 104 are performed on various drugs and administered samples, and the change in the binding property of the specific protein to the carrier protein is analyzed, thereby being involved in the disease mechanism of the specific protein. Can be estimated.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

[Experimental method]
In this example, the experiment was performed according to the flowchart shown in FIG. FIG. 4 shows a schematic diagram of the experimental operation of this example.

  As shown in FIGS. 3 and 4, first, a substance that specifically binds to the target carrier protein is prepared (step 1 in FIG. 2 (indicated as S1 in the figure, and the following steps are also indicated in the same manner)). ), Carrier protein was immobilized on one surface of the flat support 301 (step 2). The support 301 is made of glass and is combined with the chamber 302.

  Step 2 was performed as follows. The chamber 302 is a sheet made of polydimethylsiloxane (PDMS). In addition, the chamber 302 has a concave groove 303 formed in a substantially central portion with a length of 18 mm, a width of 15 mm, and a depth of 0.35 mm. Since the chamber 302 is made of PDMS, it is adsorbed when placed on one main surface of the glass support 301. When the chamber 302 is adsorbed to one main surface of the support 301, a reaction tank is formed between the concave groove 303 and one main surface of the support 301. In addition, a pair of inflow ports 304 are provided at the corners of the groove 303 in the chamber 302 for taking samples and reagents into and out of the reaction tank.

  In a state where the reaction tank is formed in this way, a reagent containing a substance that specifically binds to the carrier protein is injected into the reaction tank from one inflow port 304 using the micropipettor 305. In this state, the support 301 assembled with the chamber 302 is placed inside the sealed container 307 together with the water droplets 306. The inside of the sealed container 307 can maintain a predetermined humidity for a long time by the water droplets 306. Thereby, evaporation of the solution injected into the reaction vessel can be prevented.

  The sealed container 307 is disposed inside the gas phase incubator 308 maintained at a desired temperature. As a result, the reagent and the support 301 are incubated in the reaction vessel. Next, the support 301 is immersed in the cleaning solution in the cleaning container 309 with the chamber 302 removed. In this state, the cleaning container 309 was shaken to remove unnecessary components such as substances that were not bonded to the support 301. In this manner, a substance that specifically binds to the carrier protein could be immobilized on one main surface of the support 301.

  Next, separately prepared sample 311 to be analyzed (step 3) is brought into contact with one main surface of the support 301 to which the chamber 302 is newly attached (step 4). At this time, the sample 311 to be analyzed is injected into the reaction vessel from the inlet 304 using the micropipette 305. Thereafter, the support 301 was placed inside the sealed container 307, and incubated in the gas phase incubator 308 while being placed in the sealed container 307 (step 5). After incubation under predetermined conditions (temperature, time, and humidity), the chamber 302 was removed, and the support 301 was washed with the washing container 309 (step 6). By this operation, the carrier protein contained in the sample 311 to be analyzed is captured by the support 311.

  Next, the chamber 302 was attached again to one main surface of the support 311, and the elution reagent 312 was injected into the reaction tank from the inflow port 304 using the micropipette 305. The elution reagent 312 was injected into the reaction tank and placed inside the sealed container 307, and incubated in the gas phase incubator 308 while placed in the sealed container 307. As a result, the carrier protein is eluted from one main surface of the support 311 inside the reaction vessel (step 7).

  Next, the solution inside the reaction vessel was recovered from the inlet 304 using the micropipette 305. The collected solution was transferred into a microtube, and then protease reagent 313 was added to digest the carrier protein contained in the solution with protease (step 8). By Step 8, the carrier protein contained in the collected solution is decomposed into peptide fragments, and the molecules that interacted with the carrier protein are released into the solution.

  Separately, a polyclonal antibody using a carrier protein as an antigen was prepared (step 9), and a support 314 on which the obtained polyclonal antibody was immobilized was prepared (step 10). The solution after the treatment in Step 8 was brought into contact with the support 314 to which the chamber 302 was attached (Step 11). Specifically, the solution after the process of Step 8 was injected into the reaction vessel from the inlet 304 using the micropipette 305. Then, the solution after the treatment in Step 8 was poured into the reaction vessel and placed inside the sealed container 307, and incubated in the gas phase incubator 308 while placed in the sealed container 307 (Step 12).

  After completion of the incubation, the solution inside the reaction vessel was recovered from the inlet 304 using the micropipette 305 (step 13). At this time, since the peptide fragment derived from the carrier protein is captured by the polyclonal antibody immobilized on the support 314, the solution collected in Step 13 contains almost no peptide fragment derived from the carrier protein. The molecule that interacted with is contained as a main component. Next, the recovered solution was desalted with a commercially available desalting device 315 (step 14), and then measured with a mass spectrometer 316 (step 15). A database search using analysis software identified the molecules that interacted with the carrier protein.

[Example 1]
In this experimental example, in accordance with the experimental method described above, mouse-derived anti-human IL-8 immunoglobulin (anti-IL-8 IgG) is used as a carrier protein model, and recombinant human IL- is used as a model of a molecule that interacts with the carrier protein. 8 was used. Further, the complex of anti-IL-8 IgG and IL-8 was recovered by a support on which Protein G that specifically binds to a carrier protein (anti-IL-8 IgG) was immobilized. As the protease digestion treatment, trypsin digestion treatment was performed. Peptides derived from anti-IL-8 IgG were removed by a support on which an anti-mouse IgG polyclonal antibody was immobilized. Then, human IL-8 was identified by mass spectrometry.

[Immobilization of capture molecules on support]
In this experiment, ProteoChip (Type A, Proteogen) was used as the support 301. ProteoChip is a protein chip in which a protein binding reagent 'ProLinker', which is a kind of calix crown derivative, is coated on a slide glass (26 mm long x 76 mm wide), and can be immobilized on the surface by interaction with ProLinker .

  A chamber 302 for adding a sample or a reagent was previously set on the surface of the support. Protein G (10-1201, Zymed Laboratories) was diluted to 100 μg / ml using PBS (pH 7.4) containing 30% Glycerol, and 100 μl was added using a micropipetter 305. The support was placed in a sealed container 307 with water drops 306 in the corners, and incubated overnight at 37 ° C. to immobilize Protein G on the support. The chamber was removed and the support was immersed in 50 ml of wash solution (PBS containing 0.5% Tween 20, pH 7.8) and shaken for 10 minutes to remove unbound Protein G. Excess water on the support was removed with a spin dryer (2350, Waken Pharmaceutical).

[Addition and capture of protein complex to be analyzed]
A solution containing 0.1 mg / ml each of anti-IL-8 IgG (10-I78, Fitzgerald) and recombinant human IL-8 (PHP055, Serotec) was prepared using PBS (pH 7.4) containing 1% BSA. . Using a micropipette 305, 100 μl was added to the support 301 on which Protein G was immobilized. The support was placed in a sealed container 307 with a water drop 306 in the corner and incubated overnight at 37 ° C, and the complex of anti-IL-8 IgG and IL-8 was captured on the support by immobilized Protein G. . The support was immersed in 50 ml of washing solution and shaken for 10 minutes to remove unbound protein. Excess water was removed with a spin dryer.

[Elution of captured complex]
The elution reagent 312 (0.2 M Tris-HCl containing 6 M guanidine hydrochloride and 2.5 mM EDTA, pH 8.5) was added by 100 μl using a micropipettor 305. The support was placed in a sealed container 307 containing water droplets 306 at the corners and incubated at room temperature for 10 minutes to dissociate the complex bound to Protein G. The eluted reagent was collected using a micropipettor 305.

[Reductive alkylation of complex and trypsin digestion]
1 μl of 1 mg / ml DTT solution was added to the collected solution, and the solution surface was blown with nitrogen gas, followed by incubation at 37 ° C. for 3 hours. Next, 1 μl of 0.5 mg / ml iodoacetamide solution was added, the surface of the solution was blown with nitrogen gas, and the mixture was incubated at room temperature for 1 hour in the dark. After removing guanidine hydrochloride by dialysis, 1 μl of 10 ng / ml trypsin solution 313 was added and incubated overnight at 37 ° C. to fragment the complex into peptides.

[Addition of fragmented complex to polyclonal antibody-immobilized support]
A fragmented complex solution was added to the support 314 on which a goat-derived anti-mouse IgG polyclonal antibody (AF007, R & D Systems) was immobilized, and the solution was recovered after incubation at 37 ° C. overnight.

[Desalination and mass spectrometry]
The collected solution was desalted using a commercially available desalting device Zip-Tip C18 (ZTC1 8S0 08, Millipore) 315. The desalted sample is subjected to centrifugal concentration to remove the organic solvent (acetonitrile) used for desalting, and then measured with a liquid chromatograph mass spectrometer NanoFrontierL (Hitachi High-Technologies) 316. The analysis software MASCOT (Matrix Science The protein was identified by database search using

[Experimental result]
As a result of the experiment, we succeeded in identifying human IL-8, a molecule that interacts with the carrier protein. The identification score was 161, the number of hit peptides was 4, and the sequence coverage was 40%. The identification score is calculated based on the method described in the reference (Electrophoresis, 20, 3551-67, (1999)). In addition, since anti-IL-8 IgG was not detected, the peptide fragment derived from anti-IL-8 IgG was almost completely removed by treatment with support 314 immobilized with anti-mouse IgG polyclonal antibody. It was shown that. From the above results, it was shown that this method can be used as a sample preparation method for analyzing a protein interacting with a carrier protein.

[Experimental Example 2] Identification of Albumin-Binding Disease Marker This example is an experimental example for identifying a novel disease marker in a specific disease. In particular, in this example, a novel disease marker is screened from albumin binding substances.

  First, blood is collected from each of a patient group and a healthy group, and a serum sample is prepared. The obtained serum sample is added to a support on which an anti-albumin monoclonal antibody is immobilized, and the albumin component is selectively captured from the serum sample. Next, after the captured albumin component is eluted, digestion with trypsin is performed to decompose the albumin component into peptide fragments. By this treatment, the albumin binding substance is released into the solution after the treatment. Next, the solution after the treatment is added to a support on which an anti-albumin polyclonal antibody is immobilized, and the peptide fragment derived from albumin is captured by the anti-albumin polyclonal antibody. Thereafter, the components excluding the peptide fragments derived from albumin are collected and used for mass spectrometry. Albumin binding substances can be identified by mass spectrometry.

  In accordance with the above steps, an albumin binding substance in which a significant difference is observed between the patient group and the healthy subject group is identified as a candidate substance for a disease marker.

[Experimental Example 3] Evaluation of albumin binding property of drugs Some drugs improve stability in vivo by binding to albumin in the body. Therefore, by evaluating the albumin binding property of a drug candidate substance, the stability of the candidate substance in the body can be evaluated.

  Specifically, a specific drug candidate substance is added to a serum sample prepared according to a conventional method. Then, the component couple | bonded with the albumin contained in a sample is identified by the method similar to Example 2. FIG. Thereby, the albumin binding property of the reagent-agent candidate substance can be evaluated.

  On the other hand, a plurality of drug candidate substances are added to the serum sample, and similarly, components bound to albumin are identified. Thereby, it is possible to evaluate which candidate substance binds to albumin among a plurality of drug candidate substances.

It is a flowchart of the method of detecting the molecule | numerator which interacted with carrier protein to which this invention is applied. It is a schematic diagram which shows the principle of each process in the method of detecting the molecule | numerator which interacted with carrier protein to which this invention is applied. It is a flowchart of the method implemented in the Example. It is a schematic diagram of each experiment operation implemented in the Example.

Explanation of symbols

201 ... support, 202 ... substance having affinity for carrier protein, 203 ... carrier protein, 204 ... molecule interacting with carrier protein, 205 ... contaminant, 206 ... peptide fragment, 207 ... polyclonal antibody, 208 ... mass Analytical device, 301 ... support, 302 ... chamber, 303 ... groove, 304 ... inlet, 305 ... micro pipettor, 306 ... water droplet, 307 ... sealed container, 308 ... gas phase incubator, 309 ... cleaning container, 311 ... sample 312 ... Elution reagent, 313 ... Protease reagent, 314 ... Support, 315 ... Desalting device, 316 ... Mass spectrometer

Claims (13)

A method for detecting molecules interacting with a carrier protein,
Separating the carrier protein from the sample containing the carrier protein that interacted with the molecule,
Decompose carrier protein after separation,
Separate the peptide fragment derived from the carrier protein from the molecule that interacted with the carrier protein,
A method for detecting molecules bound to a carrier protein.   2. The method according to claim 1, wherein a polyclonal antibody against the carrier protein is used when separating the peptide fragment derived from the carrier protein from the molecule interacting with the carrier protein.   The method according to claim 1, wherein the molecule to be detected is a protein.   The method according to claim 1, wherein when detecting a molecule interacting with a carrier protein, the molecule is identified by mass spectrometry. A kit for separating molecules interacting with a carrier protein,
A first separation means for separating the carrier protein from a sample containing the carrier protein interacting with the molecule;
A degradation means capable of degrading the carrier protein;
A second separation means for separating the peptide fragment produced by the decomposition of the carrier protein by the decomposition means and the molecule that interacted with the carrier protein;
A kit comprising: The kit according to claim 5, wherein the second separation means has a polyclonal antibody against a carrier protein.   The kit according to claim 5, wherein the decomposing means includes a proteolytic enzyme. The kit according to claim 5, which is used for detecting a molecule that interacts with a carrier protein contained in a serum sample. An apparatus for detecting molecules interacting with a carrier protein,
A first separation means for separating the carrier protein from a sample containing the carrier protein interacting with the molecule;
Decomposition means for decomposing the carrier protein separated by the first separation means;
A second separation means for separating the peptide fragment produced by the decomposition of the carrier protein by the decomposition means and the molecule that interacted with the carrier protein;
A detection means for detecting a molecule that has been interacted with the carrier protein separated by the second separation means. The apparatus according to claim 9, wherein the second separation means has a polyclonal antibody against a carrier protein.   The apparatus according to claim 9, wherein the decomposing means includes a proteolytic enzyme.   The apparatus according to claim 9, wherein the detection means is a mass spectrometer. The apparatus according to claim 9, further comprising an arithmetic unit having a computer program for identifying the molecule based on an output from the detection means .

Images