JP2012037349A - Biomarker of cognitive function disorder disease and method for detecting cognitive function disorder disease using the biomarker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a cognitive function disorder disease including a slight cognitive function disorder and Alzheimer's disease using protein and their partial peptide with different existing amount in a non-cognitive function disorder subject and a patient with the cognitive function disorder disease, and a biomarker comprising the protein and the partial peptide for detecting the cognitive function disorder disease including the slight cognitive function disorder and Alzheimer's disease.SOLUTION: The present invention provides the biomarker for diagnosing the cognitive function disorder disease comprising at least one protein to be selected from protein of sequence numbers 1, 3, 5, 7, and amino acid sequence to be expressed by sequence numbers 2, 4, 6 which is the partial peptide of the protein and peptide fragments with five or more amino acid residues to be generated from the protein, and a diagnostic method using the same.

Description

  The present invention relates to biomarkers that are novel proteins and peptides that can be used for detection of cognitive dysfunction diseases including mild cognitive dysfunction and Alzheimer's disease, and a method for detecting cognitive dysfunction diseases using the biomarkers.

  As a means for discriminating the difference using a sample exhibiting a normal state and a non-normal state of a living body, a technique that has been generally used in an in vitro diagnostic agent is a conventional technique. The most common in vitro diagnostic agents are those that perform diagnostic tests by analyzing blood components as biomarkers. In the prior art in this field, the presence of a single specific protein in blood or a so-called peptide having a molecular weight of 10,000 or less, or the activity in the case of an enzyme protein is measured, and a normal (healthy person) sample and a disease sample are measured. It has helped diagnosis with the obvious difference. That is, measure the amount or activity of single or multiple specific proteins or peptides in a certain number of healthy subjects and samples from disease patients in advance, determine the range of abnormal values and normal values, and evaluate the samples in the same way Measurement is performed and inspection evaluation is performed depending on whether the value belongs to an abnormal value range or a normal value range.

As a specific measurement method, a specific primary antibody labeled with an enzyme that develops color when a sample is directly or diluted in advance and reacted with a substrate with a single or a plurality of specific proteins or peptides. Enzyme-linked immunosorbent assay (ELISA) or chemiluminescence assay (CLIA) that measures the color development amount of the sample using a secondary antibody and binding to the primary antibody or secondary antibody There are a radioimmunoassay (RIA; RadioImmunoassay) that uses a radioisotope, and an enzyme activity assay that measures the product by color development by directly giving a substrate when the protein is an enzyme. These methods using antibodies will be referred to as enzyme or fluorescent or radioactive substance labeled antibody methods. There is also a method for analyzing enzyme degradation products by high performance liquid chromatography (HPLC). LC-MS / MS method combining HPLC and mass spectrometer and selected reaction using this method
There is also a monitoring (SRM) / multiple reaction monitoring (MRM) method. In addition, after subjecting the sample to appropriate pretreatment, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is performed to separate the protein or peptide, and then the target protein or peptide is stained with silver, Coomassie blue, or There is also a method of measuring the concentration in a sample by performing immunostaining (Western blotting) using a corresponding antibody. In addition, there is a technique in which a biological sample is fractionated by column chromatography, and proteins and peptides contained in the fraction are analyzed by mass spectrometry. Further, there is a method of mass spectrometry using a protein chip as a pretreatment instead of column chromatography, and a method of mass spectrometry using magnetic beads as a pretreatment.

  Furthermore, the inventor binds an antibody against a protein or peptide of interest to beads (including magnetic beads), captures the protein or peptide to be measured by this, and then elutes from the beads and measures by mass spectrometry. We are developing. In addition, for the purpose of analyzing an intact protein, a method of performing mass spectrometry using the above method after digestion with trypsin or the like has been reported (see Patent Document 1). However, in any case, utilizing the properties of intact proteins, fractionation is performed as it is, or protein molecules that are specifically adsorbed are selected and analyzed by mass spectrometry.

  Cognitive dysfunction diseases such as Alzheimer's disease are increasing rapidly in Japan with the recent aging. The number was about 1.3 million in 1995, but about 1.9 million in 2005 and is expected to reach about 3 million in 2020. Alzheimer's disease is said to account for 60-90% of cognitive impairment disorders. This disease is not only losing the patient's memory, but also destroying the personality and losing the patient's social life function, so it is becoming a social problem. In Japan, Donepezil hydrochloride, an anti-acetylcholinesterase inhibitor, was approved at the end of 1999, and if it was administered early, it became possible to “delay” the decline in cognitive function with a high probability. In Alzheimer's disease, early diagnosis is the most important issue in order to increase the effects of current therapies and therapeutic drugs to be developed.

The main diagnostic criteria (DSM IV) for Alzheimer's disease by the American Psychiatric Association are shown below.
A. A variety of cognitive deficits manifested by both:
(1) Memory impairment (disability of learning new information or recalling previously learned information)
(2) one or more of the following cognitive impairments
a) Aphasia (language disorder)
b) Apraxia (disability of ability to perform movements despite no impairment of motor function)
c) Agnosia (impaired ability to recognize or identify a subject despite no sensory impairment)
d) Obstacles to ability to execute (plan, organize, order, abstract)
B. Cognitive deficits in criteria A (1) and A (2) each cause a significant impairment in social or occupational function and show a significant decline from the pre-morbid function level (Non-patent Document 1).

  There are various diseases associated with Alzheimer disease (AD). Since dementia such as AD gradually shows a decline in cognitive function, there is a state that should be called a precursor state of dementia. Such a condition is called mild cognitive impairment (MCI). According to US data, 10-15% of MCIs who visited a forgetfulness outpatient clinic are transferred to AD in 10-15% per year and 4 years. Most of the precursor states of AD are included in amnestic MCI. According to the current definition, MCI hears complaints about cognitive decline, but does not interfere with basic daily life. Frontotemporal dementia (FTD) is characterized by cognitive decline and behavior that goes along my way without worrying about the surroundings, as opposed to AD that tries to adjust to the surroundings. FTD includes Pick's disease, which histologically shows the presence of Pick spheres in the cerebral cortex. Lewy body dementia (DLB) is characterized by progressive memory impairment and visual cognitive impairment such as hallucinations. Diagnosis based on clinical symptoms is DLB, with 10-30% of dementia being the second most common degenerative dementia disease in the elderly after Alzheimer-type dementia (AD). Histologically, it is characterized by the presence of Lewy bodies in the cerebrum. Since FTD and DLB are dementia with dementia, they are also called dementia-type neurological diseases (Non-patent Document 1).

  In the present invention, when collectively referred to as cognitive dysfunction diseases, MCI, AD, and dementia-type neurological diseases are included.

  Tests widely used in the diagnosis of dementia are based on the results of interviews with subjects using the revised Hasegawa Intelligence Scale (HDS-R) and MMSE (Mini-Mental State Examination). . HDS was revised in 1991 and is now called HDS-R. It consists of nine questions and tests for orientation, memorization, computing power, memory / recollection and common sense. Defects are suspected of being 30 out of 30 and below 23. MMSE was devised in the United States for the diagnosis of dementia and covers orientation, memory, computational power, linguistic skills, and graphic skills. It consists of 11 questions with a maximum score of 30. Like HDS-R, it has a score of 23 or less and is suspected of having dementia. The results of both tests agree well with the proportions. These interrogation methods are used only for screening purposes, do not lead to a definitive diagnosis, and neither HDS-R nor MMSE are used for severity classification (Non-patent Document 1).

  The diagnostic imaging includes CT / MRI for cerebral morphological abnormalities such as brain atrophy and cerebral cerebral ventricular enlargement, cerebral blood flow scintigraphy (SPECT) for cerebral blood flow, and oxygen consumption / glucose consumption. There is positron tomography (PET). SPECT and PET are nuclear medicine methods that can detect abnormalities before morphological abnormalities occur (Non-patent Document 1). However, since these image diagnosis requires special equipment, it has a drawback that it cannot be performed in all medical institutions. In addition, judgment may be different depending on the doctor who views the image, and the objectivity is lacking.

  Thus, the diagnosis of dementia, including AD, currently depends on methods that are not objective and require the use of expensive devices, and screening for disease detection is impossible. . If a biomarker is found here that allows objective diagnosis using easily obtained patient samples such as blood (including serum and plasma), screening will present the most important issues It becomes possible to detect early cognitive dysfunction diseases. The present invention provides such a novel biomarker and a method for detecting a cognitive dysfunction disease using the biomarker.

JP 2004-333274 A JP 2006-308533 A

Nakano Imabari, Mizusawa Hidehiro Editing: Alzheimer's Disease, 2004, Nagai Shoten N. Benkirane et al., J. Biol. Chem. Vol. 268, 26279-26285, 1993

  The present invention relates to non-demented control subjects (non-demented controls, including healthy individuals, who may be affected by any disease but who are not affected by cognitive impairment disease, ie, healthy subjects, hereinafter abbreviated as NDC). ) And a cognitive dysfunction disease patient, a method for detecting cognitive dysfunction diseases including mild cognitive dysfunction and Alzheimer's disease using a protein having different presence or abundance and a partial peptide thereof, and further comprising the protein and the protein It is intended to provide a biomarker for detecting mild cognitive dysfunction consisting of partial peptides and cognitive dysfunction diseases including Alzheimer's disease.

  The inventor has intensively studied a method for detecting a cognitive dysfunction disease, and found in the serum a peptide capable of detecting a cognitive dysfunction disease including mild cognitive dysfunction and Alzheimer's disease. The peptide found in the present invention has significance as a biomarker when it is detected not only in serum but also in other biological samples such as blood, plasma, cerebrospinal fluid, and urine. At the same time, proteins or peptides that are the origin of these peptides (hereinafter referred to as intact proteins or peptides) also have significance as biomarkers.

Specifically, the present inventor has prepared Gamma-aminobutyric acid receptor subunit alpha-4 consisting of the amino acid sequence represented by SEQ ID NO: 1 and Gamma-aminobutyric acid receptor subunit alpha- consisting of the amino acid sequence represented by SEQ ID NO: 3. 6. Gelsolin (isoform 1) consisting of the amino acid sequence represented by SEQ ID NO: 5, Gelsolin consisting of the amino acid sequence represented by SEQ ID NO: 7
It has been found that at least one protein or peptide selected from (isoform 2) or a peptide fragment having 5 or more amino acid residues generated from the protein or peptide can be used as a biomarker for diagnosis of cognitive dysfunction disease.

  Furthermore, Gamma-aminobutyric acid receptor-derived peptide GBRA (VS +) consisting of the amino acid sequence represented by SEQ ID NO: 2, Gamma-aminobutyric acid receptor-derived peptide GBRA (VS-) consisting of the amino acid sequence represented by SEQ ID NO: 4, and It has been found that the Gelsolin-derived peptide GELS consisting of the amino acid sequence represented by SEQ ID NO: 6 can be used as a biomarker for diagnosis of cognitive impairment disorders.

  Furthermore, the present inventors have used these proteins and peptides or peptide fragments by using two-dimensional liquid chromatography (2D-LC) MALDI TOF-MS (mass spectrometry) and immunoMS methods, so that many proteins can be obtained at once. In addition, the present inventors have succeeded in measuring peptides or peptide fragments and have completed the present invention.

That is, the aspects of the present invention are as follows.
[1] Gamma-aminobutyric acid consisting of the amino acid sequence represented by SEQ ID NO: 1
receptor subunit alpha-4, Gamma-aminobutyric consisting of the amino acid sequence represented by SEQ ID NO: 3
acid receptor subunit alpha-6, Gelsolin consisting of the amino acid sequence represented by SEQ ID NO: 5
(isoform 1), at least one protein or peptide selected from the group consisting of Gelsolin (isoform 2) consisting of the amino acid sequence represented by SEQ ID NO: 7, or a peptide having 5 or more amino acid residues generated from the protein or the peptide A biomarker for diagnosis of cognitive dysfunction diseases consisting of fragments.

[2] Gamma-aminobutyric acid consisting of the amino acid sequence represented by SEQ ID NO: 2
Selected from receptor-derived peptide GBRA (VS +), Gamma-aminobutyric acid receptor-derived peptide GBRA (VS-) consisting of the amino acid sequence represented by SEQ ID NO: 4, and Gelsolin-derived peptide GELS consisting of the amino acid sequence represented by SEQ ID NO: 6 Biomarker for diagnosis of cognitive impairment disorders.

[3] Compared to a biological sample of a subject not suffering from mental illness in a biological sample of a patient with a cognitive dysfunction disorder selected from peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2, 4, and 6 Cognitive dysfunction disease biomarkers appearing or increasing.

[4] Appears or increases in a biological sample of an Alzheimer's disease patient selected from peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2, 4, and 6 as compared to a biological sample of a patient with non-demented neurological disease Alzheimer's disease biomarker.

[5] A method for detecting a cognitive dysfunction disease, comprising measuring at least one biomarker for diagnosis of a cognitive dysfunction disease according to any one of [1] to [4] in a biological sample.

[6] Any one of [1] to [4], wherein detection is performed by immunoblotting or Western blotting, enzyme or fluorescent or radioactive substance labeled antibody method, mass spectrometry, immunoMS method or surface plasmon resonance method The detection method of cognitive dysfunction disease of claim | item.

[7] A kit for detecting a cognitive dysfunction disease for measuring at least one biomarker according to any one of [1] to [4].

[8] A kit for detecting a cognitive dysfunction disease comprising an antibody or aptamer to at least one biomarker according to any one of [1] to [4].

  According to the present invention, Gamma-aminobutyric acid receptor-derived peptide GBRA (VS +) consisting of the amino acid sequence represented by SEQ ID NO: 2 in a biological sample derived from a subject, Gamma-aminobutyric consisting of the amino acid sequence represented by SEQ ID NO: 4 Kind and amount of at least one peptide selected from the group consisting of the acid receptor-derived peptide GBRA (VS-) and the Gelsolin-derived peptide GELS consisting of the amino acid sequence represented by SEQ ID NO: 6 and the amino acid sequence represented by SEQ ID NO: 2 It is possible to diagnose whether or not the subject suffers from a cognitive dysfunction disease. Further, by measuring the amount of at least one peptide selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 6 Alzheimer's disease can be diagnosed when it increases compared to the biological sample of a patient with non-demented neurological disease.

  The present invention also provides a diagnostic system that is both extremely accurate and specific. According to the present invention, it becomes possible for the first time to make a highly accurate diagnosis for a cognitive dysfunction disease for which there is no specific test method for biological samples such as blood. Furthermore, the biomarker of the present invention is highly useful in determining drug effects.

Cluster diagram. A collection of points existing in the range surrounded by (A) indicates the retention time (Retention Time) and m / z of the Marker A mass peak detected in each sample group of AD, MCI, and NDC. Example of difference analysis result, Marker A. MarkerA is a Gamma-aminobutyric acid receptor subunit alpha-derived peptide GBRA (VS +) as shown in the amino acid sequence obtained as a result of MS / MS analysis in FIG. Figure 2 shows a comparison between AD, MCI, and NDC. AD-A is AD for Facility A, AD-B is AD for Facility B, MCI-A is MCI for Facility A, MCI-B is MCI for Facility B, NDC-A is NDC for Facility A, NDC-C is Facility C NDC. The MS / MS spectrum figure by the TOF / TOF type | mold mass spectrometer of Marker A (sequence number 2 GBRA (VS +)). The upper part of FIG. 3 shows the amino acid sequence of GBRA (VS +) and b ions and y ions appearing in the MS / MS spectrum. Cluster diagram. The collection of points in the range surrounded by (B) indicates the retention time (Retention Time) and m / z of the GBRA (VS-) mass peak detected in each sample group of AD, MCI, and NDC . Results of GBRA (VS-) difference analysis. Comparison between AD, MCI and NDC. The disease name and facility display are the same as in FIG. Cluster diagram. A collection of points existing in the range surrounded by (C) indicates the retention time (Retention Time) and m / z of the GELS mass peak detected in each sample group of AD, MCI, and NDC. Results of GELS difference analysis. Comparison between AD, MCI and NDC. The disease name and facility display are the same as in FIG.

  The present invention detects the type and amount of an intact protein and / or its partial peptide and simultaneously measures the variation of the type and amount of the intact protein and its partial peptide when the subject suffers from a cognitive impairment disease. This is a method for diagnosing whether or not a subject suffers from a cognitive dysfunction disease. Here, the peptide generally refers to a peptide in which amino acids having a molecular weight of 10,000 or less are linked, or the number of amino acid residues is from several to about 50 or less. In the present invention, a partial peptide of an intact protein can be used as a biomarker for detecting a cognitive dysfunction disease. In the case of a partial peptide, a peptide having a partial amino acid sequence of the amino acid sequence of the intact protein. In this case, the molecular weight is 10,000 or less. In the present invention, the intact peptide partial peptide refers to a peptide having a partial amino acid sequence of the amino acid sequence of the intact protein, and is generated as a partial peptide in the expression synthesis process by transcription and translation; After being synthesized as an intact protein, it may be digested and decomposed in vivo to produce a digested degradation product peptide. This is because protein synthesis and control mechanisms are deregulated when the living body is in a state other than normal, such as a cognitive impairment disorder. That is, the present invention determines whether a subject is in a normal state or suffers from a cognitive dysfunction disease by using expression synthesis and / or digestion degradation of protein in vivo as an index, and also suffers from a cognitive dysfunction disease. It is also a method for evaluating and discriminating the degree of progression of the disease in the case of In the present invention, the detection of a cognitive dysfunction disease refers to evaluation discrimination, that is, diagnosis, whether or not a subject suffers from a cognitive dysfunction disease. It may also include an assessment of the risk that the subject will suffer from more severe cognitive impairment.

In the method of the present invention, as an intact protein that can be used as a biomarker for detecting a cognitive impairment disorder, specifically, Gamma-aminobutyric acid receptor subunit alpha-4 consisting of the amino acid sequence represented by SEQ ID NO: 1, It consists of Gamma-aminobutyric acid receptor subunit alpha-6 consisting of the amino acid sequence represented by SEQ ID NO: 3, Gelsolin (isoform 1) consisting of the amino acid sequence represented by SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 7. Gelsolin
(isoform 2), and peptide fragments having 5 or more amino acid residues, which are partial peptides of these intact proteins, can be used for the same purpose.

  In addition, as a partial peptide that can be used as a biomarker for detection of cognitive dysfunction disease, Gamma-aminobutyric acid receptor-derived peptide GBRA (VS +) consisting of the amino acid sequence represented by SEQ ID NO: 2 and amino acid represented by SEQ ID NO: 4 Examples include Gamma-aminobutyric acid receptor-derived peptide GBRA (VS-) consisting of the sequence, and Gelsolin-derived peptide GELS consisting of the amino acid sequence represented by SEQ ID NO: 6. In the present invention, the above intact protein and peptide are used as a marker. The amino acid sequence represented by SEQ ID NOs: 1 to 3 consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added. Proteins and peptides are also included, and these proteins or peptides can also be used as biomarkers in the methods of the invention. Here, “one or several” means “one or three”, “one or two” or “one”. Furthermore, these peptides that can be used as biomarkers for detecting cognitive dysfunction diseases also include peptide fragments having 5 or more amino acid residues generated from the amino acid sequences represented by SEQ ID NOs: 1 to 21. The reason why the peptide fragment is 5 or more amino acid residues is due to the following description in Non-Patent Document 2. That is, the peptide CGGGERA in which R is replaced with K for the amino acid residue sequence IRGERA at the C-terminus (130-135) of histone H3 and IR is deleted, and the peptide CGGGERA in which CGG is bound to GERA is used instead of the peptide IRGERA. It was reported that it was recognized by the antibody obtained. This indicates that antigenic recognition is made by a peptide consisting of 4 or more amino acid residues. In the present invention, in order to have generality other than the C-terminal of histone H3, the number of residues is increased by 1 to 5 or more, but such low molecular peptides are also targeted. It is important when using a method for detection and fractionation using immunological techniques such as immunoblotting, ELISA, and immunoMS.

  A sugar chain may be added to an intact protein or a partial peptide thereof. Proteins and partial peptides to which these sugar chains are added can also be used as biomarkers for detecting cognitive impairment disorders.

  In the present invention, the biomarker may be quantified or the presence or absence may be determined by qualitative.

  As a method for separating biomarkers in a biological sample such as serum in the present invention, two-dimensional electrophoresis or two-dimensional chromatography can be used. The two types of chromatography in this case may be selected from known chromatography such as ion exchange chromatography, reverse phase chromatography, gel filtration chromatography and the like. It can also be quantified by the SRM / MRM method using the LC-MS / MS method. Furthermore, an immunoMS that binds an antibody against the protein or peptide of interest to the beads (including magnetic beads) developed by the inventor, captures the protein or peptide to be measured, and then elutes from the bead and measures by mass spectrometry By using this method, the presence or amount of the target protein, protein fragment, or peptide can be easily evaluated without using two-dimensional electrophoresis or chromatography.

  According to the method of the present invention, a subject's cognitive dysfunction can be evaluated at a mild stage, which is useful for preventive medicine. Furthermore, when psychotherapy or pharmacotherapy is performed on a patient suffering from a cognitive dysfunction disease, if the progression of the disorder is suppressed, it is also reflected in the amount of protein / partial peptide in a biological sample such as serum. By measuring this, it is possible to evaluate and determine the therapeutic effect.

  The type and amount of protein in the biological sample can be measured by various methods. When a target protein (including a protein fragment and a partial peptide) has been identified and an antibody against the protein (primary antibody) has been obtained, the following method can be used.

1. Immunoblot method The simplest method. Prepare test serum diluted in several stages, drop a certain amount (around 1 microliter) onto a suitable membrane such as nitrocellulose membrane, and air dry. After treatment with a blocking solution containing a protein such as BSA, washing and reacting with the primary antibody, and after washing, a labeled secondary antibody for detecting the primary antibody is reacted. After washing the membrane, the label is visualized and the concentration is measured.

2. Western blotting After one- or two-dimensional gel electrophoresis including isoelectric point or SDS-PAGE, the separated protein is temporarily transferred to a suitable membrane such as PVDF membrane and labeled with the primary antibody. Using the prepared secondary antibody, the abundance of the target protein is measured in the same manner as in the immunoblotting method described above.

3. ELISA method An antibody against a protein or its partial peptide is bound to a carrier such as a microtiter plate that has been subjected to special chemical modification in advance. After serial dilution of the sample, an appropriate amount is added to the microtiter plate to which the antibody is bound and incubated. . Thereafter, washing is performed to remove uncaptured proteins and partial peptides. Next, a secondary antibody conjugated with a fluorescent or chemiluminescent substance or enzyme is added and incubated. For detection, evaluation is performed by adding each substrate and then measuring visible light by fluorescent or chemiluminescent substances or enzymatic reactions. A substance capable of binding to a protein or a partial peptide thereof may be used instead of an antibody. For example, an aptamer or the like can be used.

Furthermore, although a method (refer patent document 2) is illustrated below, it is not limited to them.
4). Method using microarray (microchip) A microarray is a generic term for devices in which substances that can be bound to a substance to be measured are aligned (array) immobilized on a carrier (substrate). In the case of the present invention, antibodies or aptamers against proteins and partial peptides may be used after being aligned and immobilized. For measurement, add a biological sample to the immobilized antibody, etc., bind the protein or partial peptide to be measured on the microarray, and then add the secondary antibody to which the fluorescent or chemiluminescent substance or enzyme is bound. Incubate. Detection may be performed by adding each substrate and then measuring the fluorescent or chemiluminescent substance or visible light from the enzyme reaction.

5. Mass Spectrometry In mass spectrometry, for example, an antibody against a specific protein and its partial peptide is bound to microbeads or substrates (protein chips) that have been specially modified in advance. The microbead may be a magnetic bead. Any material can be used for the substrate. The antibodies to be used are (1) an antibody that recognizes only the full length of a specific protein, (2) an antibody that recognizes only a partial peptide, (3) an antibody that recognizes both a specific protein and its partial peptide, or A combination of (1) and (2), (1) and (3), or (2) and (3) may be used. A sample is serially diluted with a stock solution or a buffer solution, and an appropriate amount thereof is added to a microbead or a substrate to which an antibody is bound, and incubated. Thereafter, washing is performed to remove uncaptured proteins and partial peptides. Then, the proteins and partial peptides captured on the microbeads or the substrate are analyzed by mass spectrometry using MALDI-TOF-MS, SELDI-TOF-MS, etc. Measure peak intensity. A certain amount of an appropriate internal standard substance is added to the original biological sample, its peak intensity is measured, and the ratio to the peak intensity of the target substance is determined to obtain the concentration in the original biological sample I can know. This method is called immunoMS method. In addition, the sample can be diluted with a stock solution or a buffer solution or a part of the protein can be removed, and then separated by HPLC and quantified by mass spectrometry using an electrospray ionization (ESI) method. At that time, the concentration in the data can be known by absolute quantification by SRM / MRM method using an isotope-labeled internal standard peptide.

  In addition to the above methods, proteins and partial peptides can be analyzed by a method using two-dimensional electrophoresis, a method using surface plasmon resonance, or the like.

  The present invention also includes a method in which a biological sample collected from a subject is subjected to two-dimensional electrophoresis or surface plasmon resonance, and a cognitive impairment disorder is detected using the presence or absence or amount of the biomarker as an index.

  2D liquid chromatography (2D-LC) MALDI TOF-MS searches for cognitive impairment disease diagnostic marker peptides

(1) Serum sample Hereinafter, the parentheses are abbreviations.
It consists of 24 AD (Alzheimer's disease), 25 MCI (mild cognitive impairment) and 14 NDC (subjects who do not suffer from cognitive impairment, ie, healthy subjects). Each disease sample was further classified by blood collection facility group. That is, AD is classified into two groups of facility A and facility B, MCI is classified into two groups of facility A and facility B, and NDC is classified into two groups of facility A and facility C. Serum samples classified by group were mixed in equal amounts and used for testing.

(2) Method 475 μl of 0.1% trifluoroacetic acid (TFA) was added to each 25 μl of serum and boiled at 100 ° C. for 15 minutes. Thereafter, in order to recover a peptide having a molecular weight of 10,000 or less, ultrafiltration was performed using Millipore YM-10. Next, analysis by the 2D-LC MALDI TOF-MS method was performed as follows. That is, samples collected by ultrafiltration were fractionated into a total of 1,146 per sample using two-dimensional HPLC (SCX cation exchange and C18 reverse phase column). The SCX cation exchange column fractionated into 6 fractions: SCX 1 passed through, SCX 2 eluted at 10% salt, SCX 3 eluted at 20% salt, SCX 4 is the fraction eluted at 30% salt concentration, SCX
5 is a fraction eluted at a salt concentration of 50%, and SCX 6 is a fraction eluted at a salt concentration of 100%. Six fractions fractionated by SCX were fractionated into 191 fractions using a C18 reverse phase column. All samples are MALDI target plate (MTP AnchorChip (TM) 600/384) for MALDI TOF / TOF mass spectrometer (ultraflex TOF / TOF, BRUKER DALTONICS) using an online spotting robot (AccuSpot, SHIMADZU) The wells on the plate (BRUKER DALTONICS) were spotted and co-crystallized while mixing with the matrix solution (α-cyano-hydroxycinnamic acid, α-CHCA). After mounting on ultraflex TOF / TOF, laser was irradiated and the mass and peak area at that mass (including the normalization process described below) were automatically measured in reflectron mode. The peak area was normalized with 250 fmol bradykinin 1-7 for each well previously added to the matrix solution to obtain an Area value. That is, bradykinin the peak area at a specific mass of the sample
The value divided by the peak area obtained from 1-7 250 fmol was defined as the Area value. This Area value corresponds to 25 μl of sample serum. Peptides with differences in serum abundance between groups (difference analysis) were performed using Parnassum (TM) (MCBI), a multigroup statistical analysis software developed by us. Peptides in which a difference in abundance was observed were determined by MS / MS using ultraflex TOF / TOF, and the intact protein or peptide that originated the peptide was identified.

(3) Results FIG. 1 and FIG. 2 are the results of differential analysis using Parnassum software on the data obtained by subjecting the mixed sera of each disease group to 2D-LC MALDI TOF-MS. Fig. 1 shows that the first fraction (SCX 1) was fractionated into 191 fractions using a C18 reverse phase column among 6 fractions fractionated on the first dimension SCX cation exchange column. In the spectrum of fractions measured by MALDI TOF-MS, the horizontal axis represents mass (m / z), and the vertical axis represents retention time of reverse phase column chromatography. The points in the figure are TOF-MS peaks, and the part where the points are gathered is defined as a peak derived from one peptide with the same m / z and the same retention time within the error range, and this is defined as a cluster. Show. The portion surrounded by (A) in FIG. 1 is a cluster of Marker A.

FIG. 2 shows the result of the difference analysis of Marker A. Marker
A is a Gamma-aminobutyric acid receptor-derived peptide GBRA (VS +) as shown later in FIG. Figure 2 shows a comparison between NDC, MCI and AD. It was found that Marker A increased in patients with MCI and AD than NDC. These results indicate that Marker A is useful for distinguishing patients with cognitive impairment (MCI, AD) from subjects who do not have cognitive impairment, ie healthy individuals (NDC).

FIG. 3 shows the result of MS / MS analysis of Marker A using an ultraflex TOF / TOF type mass spectrometer. A sufficient signal indicating y-ion appeared, and the amino acid sequence could be easily determined. A search by Mascot was performed on this result, and the protein or peptide that was the origin (hereinafter referred to as intact protein or peptide) was identified as Gamma-aminobutyric acid receptor subunit alpha-4 or Gamma-aminobutyric acid receptor subunit alpha-6. It was revealed that the detected peptide was the amino acid sequence VSDVEMEYT present in the topological region portion common to these two proteins.
Marker
Since no peptide fragments derived from Gamma-aminobutyric acid receptor subunit alpha other than A were detected, Marker A was Gamma-aminobutyric acid receptor subunit alpha.
It was not possible to determine whether it was derived from alpha-4 or Gamma-aminobutyric acid receptor subunit alpha-6. Therefore, SEQ ID NO: 1 shows the Gamma-aminobutyric acid receptor subunit alpha-4 protein, and SEQ ID NO: 3 shows the Gamma-aminobutyric acid receptor subunit alpha-6 protein sequence. SEQ ID NO: 2 uses GBRA as UniProt's Entry Name for Gamma-aminobutyric acid receptor subunit alpha as an abbreviation for peptide name. However, as described later, GBRA has two types of peptides, GBRA (VS +) and GBRA (VS-). For other detected peptides, UniProt's Entry Name is similarly used in the following description as an abbreviation of peptide name.

Marker
Peptides with different abundance in serum between groups including A were subjected to MS / MS with ultraflex TOF / TOF to determine the amino acid sequence and identify the intact protein or peptide. The results are shown below. For peptides other than Marker A, signals indicating y-ion and b-ion appeared sufficiently, and the amino acid sequence could be easily determined. The following amino acid sequences show two sets of sequences as one set, the first of which is the intact protein amino acid sequence and the second is the detected peptide sequence. The underlined portion of the first sequence corresponds to the detected peptide sequence. 1 represents the N-terminal.

[1] Gamma-aminobutyric acid receptor subunit alpha-4 derived peptide GBRA (VS +)
Gamma-aminobutyric acid
There are two types of peptides derived from receptor subunit alpha-4 (GBRA), GBRA (VS +) means the peptide of SEQ ID NO: 2, and the peptide GBRA (VS-) of SEQ ID NO: 4 described below is N of the peptide of SEQ ID NO: 2. It means a peptide lacking two terminal amino acid residues (VS). GBRA (VS +) of SEQ ID NO: 2 showed a discriminatory power with a higher AD value when comparing AD and MCI, and a higher MCI value when comparing MCI and NDC. (Figure 2)
Intact protein / peptide
001 MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR
051 ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGP VSDVE MEYT MDVFFR
101 QTWIDKRLKY DGPIEILRLN NMMVTKVWTP DTFFRNGKKS VSHNMTAPNK
151 LFRIMRNGTI LYTMRLTISA ECPMRLVDFP MDGHACPLKF GSYAYPKSEM
201 IYTWTKGPEK SVEVPKESSS LVQYDLIGQT VSSETIKSIT GEYIVMTVYF
251 HLRRKMGYFM IQTYIPCIMT VILSQVSFWI NKESVPARTV FGITTVLTMT
301 TLSISARHSL PKVSYATAMD WFIAVCFAFV FSALIEFAAV NYFTNIQMEK
351 AKRKTSKPPQ EVPAAPVQRE KHPEAPLQNT NANLNMRKRT NALVHSESDV
401 GNRTEVGNHS SKSSTVVQES SKGTPRSYLA SSPNPFSRAN AAETISAARA
451 LPSASPTSIR TGYMPRKASV GSASTRHVFG SRLQRIKTTV NTIGATGKLS
501 ATPPPSAPPP SGSGTSKIDK YARILFPVTF GAFNMVYWVV YLSKDTMEKS
551 ESLM (SEQ ID NO: 1)

Gamma-aminobutyric
acid receptor subunit alpha peptide GBRA (VS +)
VSDVEMEYT (SEQ ID NO: 2)

[2] Gamma-aminobutyric acid receptor subunit alpha-6 derived peptide GBRA (VS +)
Peptide GBRA (VS +) of SEQ ID NO: 2 is found in Gamma-aminobutyric acid receptor subunit alpha-4 (SEQ ID NO: 1) and Gamma-aminobutyric acid receptor subunit based on MS / MS and MASCOT database search results.
It is an amino acid sequence existing in the topological region common to alpha-6 proteins. The following is Gamma-aminobutyric as SEQ ID NO: 3.
The amino acid sequence of acid receptor subunit alpha-6 intact protein is shown.
Intact protein / peptide
001 MASSLPWLCI ILWLENALGK LEVEGNFYSE NVSRILDNLL EGYDNRLRPG
051 FGGAVTEVKT DIYVTSFGP V SDVEMEYT MD VFFRQTWTDE RLKFGGPTEI
101 LSLNNLMVSK IWTPDTFFRN GKKSIAHNMT TPNKLFRIMQ NGTILYTMRL
151 TINADCPMRL VNFPMDGHAC PLKFGSYAYP KSEIIYTWKK GPLYSVEVPE
201 ESSSLLQYDL IGQTVSSETI KSNTGEYVIM TVYFHLQRKM GYFMIQIYTP
251 CIMTVILSQV SFWINKESVP ARTVFGITTV LTMTTLSISA RHSLPKVSYA
301 TAMDWFIAVC FAFVFSALIE FAAVNYFTNL QTQKAKRKAQ FAAPPTVTIS
351 KATEPLEAEI VLHPDSKYHL KKRITSLSLP IVSSSEANKV LTRAPILQST
401 PVTPPPLSPA FGGTSKIDQY SRILFPVAFA GFNLVYWVVY LSKDTMEVSS
451 SVE (SEQ ID NO: 3)

As a precaution, the sequence of GBRA (VS +) is shown below.
Gamma-aminobutyric
acid receptor subunit alpha peptide GBRA (VS +)
VSDVEMEYT (SEQ ID NO: 2)

[3] Gamma-aminobutyric acid receptor subunit alpha-4 derived peptide GBRA (VS-)
The GBRA (VS-) of SEQ ID NO: 4 forms the cluster shown in Fig. 4 (B) by Parnassum clustering. As a result of the difference analysis, AD and MCI are higher than NDC. It has been found useful for distinguishing between patients with dysfunctional disease and subjects who do not suffer from cognitive dysfunction disease, that is, healthy subjects. (Figure 5)
Intact protein / peptide
001 MVSAKKVPAI ALSAGVSFAL LRFLCLAVCL NESPGQNQKE EKLCTENFTR
051 ILDSLLDGYD NRLRPGFGGP VTEVKTDIYV TSFGPVS DVE MEYT MDVFFR
101 QTWIDKRLKY DGPIEILRLN NMMVTKVWTP DTFFRNGKKS VSHNMTAPNK
151 LFRIMRNGTI LYTMRLTISA ECPMRLVDFP MDGHACPLKF GSYAYPKSEM
201 IYTWTKGPEK SVEVPKESSS LVQYDLIGQT VSSETIKSIT GEYIVMTVYF
251 HLRRKMGYFM IQTYIPCIMT VILSQVSFWI NKESVPARTV FGITTVLTMT
301 TLSISARHSL PKVSYATAMD WFIAVCFAFV FSALIEFAAV NYFTNIQMEK
351 AKRKTSKPPQ EVPAAPVQRE KHPEAPLQNT NANLNMRKRT NALVHSESDV
401 GNRTEVGNHS SKSSTVVQES SKGTPRSYLA SSPNPFSRAN AAETISAARA
451 LPSASPTSIR TGYMPRKASV GSASTRHVFG SRLQRIKTTV NTIGATGKLS
501 ATPPPSAPPP SGSGTSKIDK YARILFPVTF GAFNMVYWVV YLSKDTMEKS
551 ESLM (SEQ ID NO: 1)

Gamma-aminobutyric
acid receptor subunit alpha peptide GBRA (VS-)
DVEMEYT (SEQ ID NO: 4)

[4] Gamma-aminobutyric acid receptor subunit alpha-6 derived peptide GBRA (VS-)
Peptide GBRA (VS-) of SEQ ID NO: 4 is obtained from the results of MS / MS and MASCOT database search, and Gamma-aminobutyric acid receptor subunit alpha-4 protein (SEQ ID NO: 1) and Gamma-aminobutyric
It is an amino acid sequence present in the topological region common to the acid receptor subunit alpha-6 protein. The amino acid sequence of Gamma-aminobutyric acid receptor subunit alpha-6 intact protein is shown below as SEQ ID NO: 3.
Intact protein / peptide
001 MASSLPWLCI ILWLENALGK LEVEGNFYSE NVSRILDNLL EGYDNRLRPG
051 FGGAVTEVKT DIYVTSFGPV S DVEMEYT MD VFFRQTWTDE RLKFGGPTEI
101 LSLNNLMVSK IWTPDTFFRN GKKSIAHNMT TPNKLFRIMQ NGTILYTMRL
151 TINADCPMRL VNFPMDGHAC PLKFGSYAYP KSEIIYTWKK GPLYSVEVPE
201 ESSSLLQYDL IGQTVSSETI KSNTGEYVIM TVYFHLQRKM GYFMIQIYTP
251 CIMTVILSQV SFWINKESVP ARTVFGITTV LTMTTLSISA RHSLPKVSYA
301 TAMDWFIAVC FAFVFSALIE FAAVNYFTNL QTQKAKRKAQ FAAPPTVTIS
351 KATEPLEAEI VLHPDSKYHL KKRITSLSLP IVSSSEANKV LTRAPILQST
401 PVTPPPLSPA FGGTSKIDQY SRILFPVAFA GFNLVYWVVY LSKDTMEVSS
451 SVE (SEQ ID NO: 3)

As a precaution, the sequence of GBRA (VS-) is shown below.
Gamma-aminobutyric
acid receptor subunit alpha peptide GBRA (VS-)
DVEMEYT (SEQ ID NO: 4)

[5] Gelsolin (isoform 1) -derived peptide GELS
Gelsolin-derived peptide GELS (SEQ ID NO: 6) forms a cluster shown in Fig. 6 (C) by Parnassum clustering. As a result of difference analysis, AD is higher than MCI and NDC, and Alzheimer type recognition It has been found useful for distinguishing symptomatic patients from subjects who do not suffer from cognitive dysfunction disease, that is, healthy subjects as well as patients with mild cognitive dysfunction disease. (Fig. 7)
Intact protein / peptide
001 MAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP QGRVPEARPN
051 SMVVEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF TGDAYVILKT
101 VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN GRAVQHREVQ
151 GFESATFLGY FKSGLKYKKG GVASGFKHVV PNEVVVQRLF QVKGRRVVRA
201 TEVPVSWESF NNGDCFILDL GNNIHQWCGS NSNRYERLKA TQVSKGIRDN
251 ERSGRARVHV SEEGTEPEAM LQVLGPKPAL PAGTEDTAKE DAANRKLAKL
301 YKVSNGAGTM SVSLVADENP FAQGALKSED CFILDHGKDG KIFVWKGKQA
351 NTEERKAALK TASDFITKMD YPKQTQVSVL PEGGETPLFK QFFKNWRDPD
401 QTD GLGLSYL SSHIANVERV PFD AATLHTS TAMAAQHGMD DDGTGQKQIW
451 RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ
501 DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG
551 TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS
601 AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA
651 YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML
701 LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG
751 FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA (SEQ ID NO: 5)

Gelsolin-derived peptide GELS
GLGLSYLSSH IANVERVPFD (SEQ ID NO: 6)

[6] Gelsolin (isoform 2) derived peptide GELS
The Gelsolin protein has two isoforms from the UniProt database. The peptide GELS of SEQ ID NO: 6 is the Gelsolin (isoform 1) protein (SEQ ID NO: 5) and Gelsolin
(isoform 2) An amino acid sequence present in a topological region common to proteins. The amino acid sequence of Gelsolin (isoform 2) intact protein is shown below as SEQ ID NO: 7.
Intact protein / peptide
001 MVVEHPEFLK AGKEPGLQIW RVEKFDLVPV PTNLYGDFFT GDAYVILKTV
051 QLRNGNLQYD LHYWLGNECS QDESGAAAIF TVQLDDYLNG RAVQHREVQG
101 FESATFLGYF KSGLKYKKGG VASGFKHVVP NEVVVQRLFQ VKGRRVVRAT
151 EVPVSWESFN NGDCFILDLG NNIHQWCGSN SNRYERLKAT QVSKGIRDNE
201 RSGRARVHVS EEGTEPEAML QVLGPKPALP AGTEDTAKED AANRKLAKLY
251 KVSNGAGTMS VSLVADENPF AQGALKSEDC FILDHGKDGK IFVWKGKQAN
301 TEERKAALKT ASDFITKMDY PKQTQVSVLP EGGETPLFKQ FFKNWRDPDQ
351 TD GLGLSYLS SHIANVERVP FD AATLHTST AMAAQHGMDD DGTGQKQIWR
401 IEGSNKVPVD PATYGQFYGG DSYIILYNYR HGGRQGQIIY NWQGAQSTQD
451 EVAASAILTA QLDEELGGTP VQSRVVQGKE PAHLMSLFGG KPMIIYKGGT
501 SREGGQTAPA STRLFQVRAN SAGATRAVEV LPKAGALNSN DAFVLKTPSA
551 AYLWVGTGAS EAEKTGAQEL LRVLRAQPVQ VAEGSEPDGF WEALGGKAAY
601 RTSPRLKDKK MDAHPPRLFA CSNKIGRFVI EEVPGELMQE DLATDDVMLL
651 DTWDQVFVWV GKDSQEEEKT EALTSAKRYI ETDPANRDRR TPITVVKQGF
701 EPPSFVGWFL GWDDDYWSVD PLDRAMAELA A (SEQ ID NO: 7)

As a precaution, the sequence of GBRA is shown below.
Gelsolin-derived peptide GELS
GLGLSYLSSH IANVERVPFD (SEQ ID NO: 6)

  Since the biomarker disclosed in the present invention can be used to detect mild cognitive dysfunction and cognitive dysfunction diseases including Alzheimer's disease, it can be applied to applications in the diagnostic field including diagnostic agents.

Claims (8)

Gamma-aminobutyric consisting of the amino acid sequence represented by SEQ ID NO: 1
acid receptor subunit alpha-4, Gamma-aminobutyric consisting of the amino acid sequence represented by SEQ ID NO: 3
acid receptor subunit alpha-6, Gelsolin consisting of the amino acid sequence represented by SEQ ID NO: 5
(isoform 1), at least one protein or peptide selected from the group consisting of Gelsolin (isoform 2) consisting of the amino acid sequence represented by SEQ ID NO: 7, or a peptide having 5 or more amino acid residues generated from the protein or the peptide A biomarker for diagnosis of cognitive dysfunction diseases consisting of fragments.   Gamma-aminobutyric acid receptor-derived peptide GBRA (VS +) consisting of the amino acid sequence represented by SEQ ID NO: 2, Gamma-aminobutyric acid receptor-derived peptide GBRA (VS-) consisting of the amino acid sequence represented by SEQ ID NO: 4, and SEQ ID NO: A biomarker for diagnosis of a cognitive dysfunction disease selected from the Gelsolin-derived peptide GELS consisting of the amino acid sequence represented by 6.   Appearance or increase in a biological sample of a patient with a cognitive dysfunction disease selected from peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2, 4, and 6 as compared to a biological sample of a subject not suffering from a mental illness Cognitive impairment disease biomarkers.   Alzheimer's disease bio, which appears or increases in a biological sample of an Alzheimer's disease patient selected from peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2, 4, and 6 as compared to a biological sample of a patient with non-demented neurological disease marker.   A method for detecting a cognitive dysfunction disease, comprising measuring a biomarker for diagnosing at least one cognitive dysfunction disease according to any one of claims 1 to 4 in a biological sample.   The recognition according to any one of claims 1 to 4, wherein the detection is performed by immunoblotting or Western blotting, enzyme or fluorescent or radioactive substance labeled antibody method, mass spectrometry, immunoMS method or surface plasmon resonance method. A method for detecting a dysfunctional disease.   A detection kit for a cognitive dysfunction disease for measuring at least one biomarker according to any one of claims 1 to 4.   A detection kit for a cognitive dysfunction disease comprising an antibody or aptamer to at least one biomarker according to any one of claims 1 to 4.