Method for analyzing neurogranin-related peptides

Abstract

A method for analyzing a neurogranin-related peptide that is included in a biological sample, said method comprising, in order: a binding step in which the biological sample is brought into contact with a carrier in a binding solution to obtain a bound body in which the neurogranin-related peptide has bound to the carrier; a washing step in which a washing solution is used to wash the bound body; an elution step in which the bound body is brought into contact with an acidic solution to obtain an eluate in which the neurogranin-related peptide has eluted into the acidic solution; and a detection step in which mass spectrometry is used to detect the neurogranin-related peptide in the eluate. The binding solution and the washing solution both include a surfactant, and the carbon number of a hydrophobic group of each surfactant is 9 to 11.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing neurogranin-related peptides. [Background technology]

[0002] Alzheimer's disease is a major cause of dementia, and the number of patients has been increasing in recent years, making research into the disease increasingly important. Aβ-related peptides, such as amyloid-β (Aβ), which are generated by cleavage of amyloid precursor protein (APP), are deeply involved in the development of Alzheimer's disease. It has been reported that immunoprecipitation and mass spectrometry can be combined to detect multiple Aβ-related peptides in blood, and that the ratio of the detected specific Aβ-related peptides is a promising blood biomarker for amyloid accumulation in the brain (Non-Patent Documents 1-2, Patent Documents 1-3).

[0003] On the other hand, various biomarkers are required to monitor the progression of Alzheimer's disease, and in addition to aluminoid accumulation, biomarkers that reflect each process of tau accumulation and neurodegeneration are required. Among these, neurogranin is one of the biomarkers for neurodegeneration, and it has been reported that it increases in the cerebrospinal fluid (CSF) of Alzheimer's disease patients (Non-Patent Documents 3 and 4). It has also been reported that in the brains of Alzheimer's disease patients, the fragmentation of neurogranin is promoted, resulting in the generation of fragment peptides (Non-Patent Document 5). Therefore, mass spectrometry of neurogranin or its fragment peptides (neurogranin-related peptides) is expected to be a means of monitoring neurodegeneration.

[0004] However, analyzing neurogranin-related peptides in cerebrospinal fluid requires collection of cerebrospinal fluid, which is undesirable from an invasive perspective. Therefore, a less invasive blood analysis, which can be collected by general testing, is desired.

[0005] However, the amount of neurogranin-related peptides present in blood is so small that they cannot be adequately detected by conventional mass spectrometry. For example, it has been reported that neurogranin or its fragment peptides could be detected by a method combining immunoprecipitation and mass spectrometry (Non-Patent Document 6). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2015 / 178398 [Patent Document 2] WO2017 / 47529 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-20980 [Non-patent literature]

[0007] [Non-Patent Document 1] Kaneko N, Nakamura A, Washimi Y, Kato T, Sakurai T, Arahata Y, Bundo M, Takeda A, Niida S, Ito K, Toba K, Tanaka K, Yanagisawa K. : Novel biomarker plasma surrogating cerebral amyloid deposition. Proc Jpn Acad Ser B Phys Biol Sci. 2014;90(9):353-364. [Non-patent document 2] Nakamura A, Kaneko N, Villemagne VL, Kato T, Doecke J, Dore V, Fowler C, Li QX, Martins R, Rowe C, Tomita T, Matsuzaki K, Ishii K, Ishii K, Arahata Y, Iwamoto S, Ito K, Tanaka K, Masters CL, Yanagisawa K. : High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018;554(7691):249–254.

Outdoor Tools3

Outdoor Tools 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0008] However, even when the analytical method of Non-Patent Document 6 is carried out, and even when the analytical method of Aβ-related peptides described in Non-Patent Document 2 is adapted for neurogranin-related peptides, the analytical sensitivity is insufficient and has not yet reached a level of practical application. Therefore, further improvement in sensitivity is required for analyzing neurogranin or its fragment peptides in blood.

[0009] An object of the present invention is to analyze neurogranin-related peptides with high sensitivity. [Means for solving the problem]

[0010] The analytical method of the first aspect of the present invention is a method for analyzing neurogranin-related peptides contained in a biological sample, and includes a first binding step of contacting the biological sample with a first carrier in a binding solution to obtain a first bound body in which the neurogranin-related peptide is bound to the first carrier; a first washing step of washing the first bound body with a first washing solution; a first elution step of contacting the first bound body with a first acidic solution to obtain a first eluate in which the neurogranin-related peptide is eluted in the first acidic solution; a neutralization step of mixing the first eluate with a neutral buffer to obtain a purified solution; and a neutralization step of contacting the purified solution with a second carrier. The method comprises, in order, a second binding step of obtaining a second conjugate in which the neurogranin-related peptide is bound to a second carrier; a second washing step of washing the second conjugate with a second washing solution; a second elution step of contacting the second conjugate with a second acidic solution to obtain a second eluate in which the neurogranin-related peptide is eluted in the second acidic solution; and a detection step of detecting the neurogranin-related peptide in the second eluate by mass spectrometry, wherein the binding solution, first washing solution, neutral buffer solution, and second washing solution all contain a surfactant, and the number of carbon atoms in the hydrophobic group of the surfactant is 9 to 11.

[0011] The analytical method of the second aspect of the present invention is a method for analyzing neurogranin-related peptides contained in a biological sample, and comprises, in order, a binding step in which the biological sample is contacted with a carrier in a binding solution to obtain a conjugate in which the neurogranin-related peptide is bound to the carrier; a washing step in which the conjugate is washed with a washing solution; an elution step in which the conjugate is contacted with an acidic solution to obtain an eluate in which the neurogranin-related peptide is eluted into the acidic solution; and a detection step in which the neurogranin-related peptide in the eluate is detected by mass spectrometry, wherein both the binding solution and the washing solution contain a surfactant, and the hydrophobic group of the surfactant has a carbon number of 9 or more and 11 or less. [Effects of the Invention]

[0012] According to the first and second aspects of the present invention, neurogranin-related peptides can be analyzed with high sensitivity. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the mass spectrum of Ng-related peptides detected by a MALDI-TOF / MS device in Reference Example 1. [Figure 2] FIG. 2 is a graph showing the relative ratio of the sensitivity (S / N) of each Example and Comparative Example to the sensitivity (S / N) of Reference Example 1 in SIL-Ng50-78. [Figure 3] FIG. 3 is a graph showing the relative ratio of the sensitivity (S / N) of each Example and Comparative Example to the sensitivity (S / N) of Reference Example 1 in Ng43-75. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. First Aspect The analytical method of the first aspect is a method for analyzing neurogranind-related peptides in a biological sample, and includes, in this order, a first binding step, a first washing step, a first elution step, a neutralization step, a second binding step, a second washing step, a second elution step, and a detection step. Each step is described in detail below.

[0015] "Neurogranin-related peptides" (hereinafter abbreviated as "Ng-related peptides") include neurogranin and fragment peptides obtained by fragmentation of neurogranin. Examples of fragment peptides include Ng50-78 (SEQ ID NO: 1) and Ng43-75 (SEQ ID NO: 2).

[0016] (1st bonding process) In the first binding step, the biological sample is contacted with the first carrier in a binding solution. For example, the binding solution, the biological sample, and the first carrier are mixed in an appropriate order. This allows the Ng-related peptide in the biological sample to bind to the first carrier, thereby obtaining a first conjugate.

[0017] Examples of biological samples include body fluids such as blood, cerebrospinal fluid, urine, bodily secretions, saliva, and sputum; and feces. Examples of blood include whole blood, plasma, and serum. The blood may be whole blood collected from an individual and then subjected to processing such as centrifugation and freezing. In the present analysis method, blood is preferably used. Blood is less invasive than cerebrospinal fluid, and is easily available as a target sample for screening in health checkups and the like.

[0018] The binding solution contains a surfactant. Preferably, the binding solution is a neutral buffer solution containing a surfactant.

[0019] Examples of neutral buffer solutions include Tris buffer, phosphate buffer, HEPES buffer, ammonium acetate buffer, etc. The pH of the neutral buffer solution is, for example, pH 6.0 or higher, preferably 6.5 or higher, and for example, pH 8.5 or lower, preferably 8.0 or lower.

[0020] The surfactant contained in the binding solution has a hydrophobic group with 9 to 11 carbon atoms. That is, the surfactant contained in the binding solution is composed of a surfactant having a hydrophobic group with 9 to 11 carbon atoms. The number of carbon atoms in the hydrophobic group is more preferably 11. By containing a surfactant with such a carbon number, nonspecific adsorption to the first binder can be suppressed.

[0021] Furthermore, from the viewpoints of suppressing protein denaturation, ease of removal, reduction of ionization interference in mass spectrometry, etc., neutral surfactants are preferred. Examples of such neutral surfactants include surfactants having maltose in the hydrophilic moiety, surfactants having trehalose in the hydrophilic moiety, and surfactants having glucose in the hydrophilic moiety.

[0022] Examples of surfactants having a hydrophobic group with 9 to 11 carbon atoms and maltose include n-nonyl-β-D-maltoside (NM), n-nonyl-β-D-thiomaltoside (NTM), n-decyl-β-D-maltoside (DM), and n-undecyl-β-D-maltoside (UDM).

[0023] Examples of surfactants having a hydrophobic group with 9 to 11 carbon atoms and trehalose include α-D-glucopyranosyl α-D-glucopyranoside monodecanoate (trehalose C10).

[0024] An example of a surfactant having a hydrophobic group with 9 to 11 carbon atoms and glucose is n-decyl-β-D-glucoside.

[0025] These surfactants can be used alone or in combination of two or more.

[0026] The critical micelle concentration (cmc) of the surfactant is, for example, 0.2 mmol / L or more, preferably 0.4 mmol / L or more, and, for example, 10 mmol / L or less, preferably 5 mmol / L or less. It is also, for example, 0.01% or more, preferably 0.02% or more, and, for example, 0.5% or less, preferably 0.3% or less. A critical micelle concentration within the above range can improve the analytical sensitivity of Ng-related peptides.

[0027] The surfactant concentration in the binding solution is, for example, 0.02% (w / v) or more, preferably 0.2% (w / v) or more, and, for example, 10% (w / v) or less, preferably 3% (w / v) or less. The surfactant concentration is, for example, 2 to 20 times the critical micelle concentration. If the surfactant concentration is within the above range, sufficient micelles are formed, ensuring the effects of the surfactant.

[0028] The first support may be any support to which an Ng-related peptide can bind, and examples thereof include antibody-immobilized supports.

[0029] The antibody immobilized on the first carrier is an antibody having an antigen-binding site capable of recognizing an Ng-related peptide (anti-Ng-related peptide antibody), and examples thereof include immunoglobulins or fragments thereof having an antigen-binding site capable of recognizing an Ng-related peptide.

[0030] Examples of immunoglobulins include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgY, IgD, and IgE. Examples of immunoglobulin fragments include F(ab')2, F(ab'), F(ab), Fd, Fv, L chains, and H chains. More specific examples include clones NG2, NG7, and EPR21152, and fragments thereof. Antibodies may be either monoclonal or polyclonal.

[0031] Examples of materials for the first support include agarose, sepharose, dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth)acrylic acid polymers, fluororesin, metal complex resin, glass, metal, and magnetic materials.

[0032] The shape of the first support may be any shape such as a sphere (including a bead shape), a plate, a needle, or an irregular shape, and may also be the wall of a flow channel in a microdevice.

[0033] Before the first binding step, a pretreatment for removing antibodies such as IgG and IgM may be carried out, if necessary.

[0034] (First cleaning process) In the first washing step, after the first binding step, the first bound body is washed with a first washing solution.

[0035] The first washing solution contains a surfactant. Preferably, the first washing solution is a neutral buffer solution containing a surfactant.

[0036] The number of carbon atoms in the hydrophobic group of the surfactant contained in the first cleaning solution is 9 or more and 11 or less, and most preferably 11. That is, the surfactant contained in the first cleaning solution is composed of a surfactant having a hydrophobic group with 9 to 11 carbon atoms. This makes it possible to effectively remove highly hydrophobic unnecessary components (such as blood proteins, lipids, and glycolipids).

[0037] The neutral buffer solution and surfactant in the first washing solution may be the same as those exemplified for the binding solution.

[0038] The surfactant concentration in the first washing solution is, for example, 0.01% (w / v) or more, preferably 0.1% (w / v) or more, and, for example, 5% (w / v) or less, preferably 2% (w / v) or less. The surfactant concentration is, for example, equal to or greater than the critical micelle concentration and equal to or less than 10 times the critical micelle concentration. If the surfactant concentration is within the above range, sufficient micelles are formed, ensuring the effects of the surfactant.

[0039] Any known washing method may be used, and preferably, washing is performed multiple times, for example, by washing with a neutral buffer containing a surfactant, followed by washing with a neutral buffer containing no surfactant.

[0040] The neutral buffer solution containing no surfactant may be the same as the neutral buffer solution exemplified as the binding solution, which can suppress foaming caused by the surfactant remaining in the first conjugate.

[0041] As the washing method, a general method may be adopted, for example, a method of stirring the carrier in a washing solution, a method of spraying the washing solution from a washing nozzle, etc. After washing with these neutral buffer solutions, washing with water may be further carried out as necessary.

[0042] (1st elution step) In the first elution step, after the first washing step, the first binder is contacted with a first acidic solution, whereby the Ng-related peptide is dissociated from the first binder, and neurogranin is eluted into the first acidic solution, resulting in a first eluate containing the Ng-related peptide.

[0043] The first acidic solution may be, for example, an acidic aqueous solution such as a glycine buffer solution or hydrochloric acid, preferably a glycine buffer solution. The pH of the first acidic solution is, for example, 3.5 or less, preferably 3.0 or less, and, for example, 0.5 or more, preferably 1.0 or more.

[0044] The first acidic solution preferably contains a surfactant. The hydrophobic group of the surfactant contained in the first acidic solution has a carbon number of 9 or more and 11 or less, and most preferably 11. That is, the surfactant contained in the first acidic solution is composed of a surfactant having a hydrophobic group with a carbon number of 9 to 11. This makes it possible to more reliably dissociate the Ng-related peptide from the first binder. Furthermore, it prevents the eluted Ng-related peptide from adhering to a container such as a test tube or a microplate. Therefore, it is possible to reliably improve the recovery rate of the Ng-related peptide.

[0045] The surfactant used in the first acidic solution may be the same as the surfactant exemplified in the binding solution, and the surfactant concentration in the first acidic solution is the same as the surfactant concentration in the first cleaning solution.

[0046] (Neutralization process) In the neutralization step, after the first elution step, the first eluate is mixed with a neutral buffer, thereby neutralizing the first eluate and obtaining a purified solution containing Ng-related peptides.

[0047] The neutral buffer solution used in the neutralization step may contain a surfactant. In particular, when the first acidic solution (and therefore the first eluate) does not contain a surfactant, it is preferable that the neutral buffer contains a surfactant. The hydrophobic group of the surfactant contained in the neutral buffer solution has 9 to 11 carbon atoms, and most preferably 11. That is, the surfactant contained in the purification solution is composed of a surfactant having a hydrophobic group with 9 to 11 carbon atoms. This makes it possible to suppress nonspecific adsorption to the second binder in the second binding step.

[0048] The neutral buffer and surfactant used in the neutralization step may be the same as those exemplified for the binding solution, and the surfactant concentration in the neutral buffer is the same as that in the first binding solution.

[0049] The pH of the purification solution is neutral, for example, pH 6.0 or higher, preferably 6.5 or higher, and for example, pH 8.5 or lower, preferably 8.0 or lower, which can improve the binding efficiency in the second binding step.

[0050] (Second bonding process) In the second binding step, after the neutralization step, the purified solution is contacted with a second carrier, whereby the Ng-related peptide in the purified solution binds to the second carrier to obtain a second conjugate.

[0051] The second support is preferably an antibody-immobilized support, and specific examples thereof include the same antibody-immobilized supports as those exemplified as the first support.

[0052] (Second cleaning process) In the second washing step, after the second binding step, the second bound body is washed with a second washing solution.

[0053] The second washing solution contains a surfactant. Preferably, the second washing solution is a neutral buffer solution containing a surfactant.

[0054] The number of carbon atoms in the hydrophobic group of the surfactant contained in the second cleaning solution is 9 or more and 11 or less, and most preferably 11. That is, the surfactant contained in the second cleaning solution is composed of a surfactant having a hydrophobic group with 9 to 11 carbon atoms. This makes it possible to effectively remove, for example, highly hydrophobic unnecessary components (such as blood proteins, lipids, and glycolipids).

[0055] The neutral buffer and surfactant used in the second washing solution may be the same as those exemplified for the binding solution, and the surfactant concentration in the second washing solution is the same as that in the first washing solution.

[0056] Any known method may be used for the washing, and specifically, the same method as the washing method exemplified in the first washing step may be carried out.

[0057] (Second elution step) In the second elution step, after the second washing step, the second binder is contacted with a second acidic solution, whereby the Ng-related peptide is dissociated from the second binder and eluted into the second acidic solution, resulting in a second eluate containing the Ng-related peptide.

[0058] Examples of the second acidic solution include the same as the first acidic solution exemplified in the first elution step, and preferably, hydrochloric acid.

[0059] The second acidic solution preferably contains a volatile organic solvent, which allows the Ng-related peptide to be efficiently dissociated from the second binder and eluted into the second acidic solution, thereby improving the recovery rate of the Ng-related peptide.

[0060] Examples of volatile organic solvents include organic solvents that are miscible with water in any ratio, such as acetonitrile, methanol, ethanol, acetone, toluene, isopropanol, hexane, butanol, cyclohexane, ethylene glycol, benzene, chloroform, acetaldehyde, triethylamine, phenol, naphthalene, formaldehyde, tetrahydrofuran, ethyl acetate, etc., and preferably acetonitrile, methanol, ethanol, acetone, isopropanol, etc. These organic solvents can be used alone or in combination of two or more.

[0061] The concentration of the volatile organic solvent in the second acidic solution is, for example, 10% (v / v) or more, preferably 25% (v / v) or more, and, for example, 90% (v / v) or less, preferably 80% (v / v) or less. When the concentration is within the above range, Ng-related peptides can be efficiently dissociated from the second carrier, and the sensitivity (S / N ratio) during mass spectrometry can be improved.

[0062] The second acidic solution preferably further contains an amino acid such as methionine. This reduces oxidation of Ng-related peptides during the time between placement in a mass spectrometer and the start of analysis, thereby improving analytical accuracy. The amino acid concentration in the second acidic solution is, for example, 0.01 mM or more, preferably 0.05 mM or more, and, for example, 5 mM or less, preferably 1 mM or less.

[0063] (Analysis process) In the analysis step, after the second elution step, the Ng-related peptides contained in the second eluate are detected by mass spectrometry.

[0064] Examples of mass spectrometry include MALDI (Matrix Assisted Laser Desorption / Ionization), ESI (Electrospray ionization), and APCI (Atmospheric Pressure Chemical Ionization). MALDI is preferred because it can be measured without liquid chromatography, thereby reducing losses due to adsorption and enabling reliable ionization of Ng-related peptides even in the presence of some contaminants.

[0065] For MALDI detection, for example, a MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometer, a MALDI-IT (matrix-assisted laser desorption ionization-ion trap) mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption ionization-ion trap-time of flight) mass spectrometer, or a MALDI-FTICR (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance) mass spectrometer may be used, operated according to a conventional method.

[0066] For detection by MALDI, the matrix is placed, for example, by dropping a matrix-containing solution onto a MALDI plate and allowing it to dry.

[0067] Examples of matrices include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, sinapic acid, 3-aminoquinoline, etc. These matrices can be used alone or in combination of two or more.

[0068] Examples of the solvent contained in the matrix include acetonitrile, trifluoroacetic acid, methanol, ethanol, water, etc. These solvents can be used alone or in combination of two or more.

[0069] The matrix concentration in the matrix-containing solvent is, for example, 0.1 mg / mL or more, preferably 0.5 mg / mL or more, and for example, 50 mg / mL or less, preferably 10 mg / mL or less.

[0070] Preferably, a matrix additive is used in combination with the matrix. Examples of the matrix additive include a phosphonic acid group-containing compound and an ammonium salt. Preferably, a phosphonic acid group-containing compound is used from the viewpoint of suppressing adverse effects on background due to residual cleaning solution. Examples of the phosphonic acid group-containing compound include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, methylenediphosphonic acid (MDPNA), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, and ethylenediaminotetraphosphonic acid.

[0071] The concentration of the matrix additive in the matrix-containing solvent is, for example, 0.01% (w / v) or more, preferably 0.1% (w / v) or more, and for example, 10% (w / v) or less, preferably 1% (w / v) or less.

[0072] This allows measurement of Ng-related peptides contained in the second eluate (and thus the biological sample). In this analytical method, by adding a specific surfactant having a hydrophobic group with 9 to 11 carbon atoms to the binding solution used in the first binding solution, the first washing solution used in the first washing step, the neutral buffer solution used in the neutralization step, and the second washing solution used in the second washing step, it is possible to highly improve the binding efficiency of Ng-related peptides to the carrier in the binding step and the recovery rate of Ng-related peptides in the elution step, and as a result, Ng-related peptides can be analyzed with extremely high sensitivity (S / N) compared to conventional analytical methods.

[0073] 2. Second Aspect In the analytical method of the first embodiment, two immunoprecipitations (affinity purifications) are performed, but a single immunoprecipitation may also be performed. In this case, the analytical method of the second embodiment comprises a first binding step (binding step), a first washing step (washing step), a second elution step (elution step), and a detection step. Each step is the same as in the first embodiment. This second embodiment also achieves the same effects as the first embodiment. The first embodiment is preferred from the viewpoint of being able to reliably analyze Ng-related peptides even when the amount of Ng-related peptides present in a biological sample is much smaller.

[0074] 3. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0075] (Item 1) An analytical method according to one embodiment is a method for analyzing neurogranin-related peptides contained in a biological sample, and includes, in order, a binding step of contacting the biological sample with a carrier in a binding solution to obtain a conjugate in which the neurogranin-related peptide is bound to the carrier; a washing step of washing the conjugate with a washing solution; an elution step of contacting the conjugate with an acidic solution to obtain an eluate in which the neurogranin-related peptide is eluted into the acidic solution; and a detection step of detecting the neurogranin-related peptide in the eluate by mass spectrometry, wherein both the binding solution and the washing solution contain a surfactant, and the number of carbon atoms in the hydrophobic group of the surfactant may be 9 or more and 11 or less.

[0076] (Item 2) In the analytical method according to item 1, the acidic solution may contain an organic solvent.

[0077] (Item 3) In the analytical method according to item 1 or 2, the biological sample may be blood.

[0078] (Item 4) In the analysis method according to any one of items 1 to 3, the mass spectrometry may be matrix-assisted laser desorption / ionization.

[0079] (Item 5) An analytical method according to one embodiment is a method for analyzing a neurogranin-related peptide contained in a biological sample, the method comprising: a first binding step of contacting the biological sample with a first carrier in a binding solution to obtain a first conjugate in which the neurogranin-related peptide is bound to the first carrier; a first washing step of washing the first conjugate with a first washing solution; a first elution step of contacting the first conjugate with a first acidic solution to obtain a first eluate in which the neurogranin-related peptide is eluted in the first acidic solution; a neutralization step of mixing the first eluate with a neutral buffer to obtain a purified solution; and a neutralization step of contacting the purified solution with a second carrier. The method includes, in order, a second binding step of obtaining a second conjugate in which the neurogranin-related peptide is bound to the second support; a second washing step of washing the second conjugate with a second washing solution; a second elution step of contacting the second conjugate with a second acidic solution to obtain a second eluate in which the neurogranin-related peptide is eluted in the second acidic solution; and a detection step of detecting the neurogranin-related peptide in the second eluate by mass spectrometry, wherein the binding solution, the first washing solution, and the second washing solution all contain a surfactant, and the number of carbon atoms in the hydrophobic group of the surfactant may be 9 or more and 11 or less.

[0080] (Item 6) In the analytical method according to item 5, the second acidic solution may contain an organic solvent.

[0081] (Item 7) In the analytical method described in item 5 or 6, the first acidic solution may contain a surfactant, and the number of carbon atoms in the hydrophobic group of the surfactant may be 9 or more and 11 or less.

[0082] (Item 8) In the analysis method according to any one of Items 5 to 7, the biological sample may be blood.

[0083] (Item 9) In the analysis method according to any one of Items 5 to 8, the mass spectrometry may be matrix-assisted laser desorption / ionization. [Example]

[0084] The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

[0085] <Reference Example 1> As Reference Example 1, Ng-related peptides were analyzed as follows, with reference to the mass spectrometry method for amyloid β described in Non-Patent Document 2.

[0086] (First binding step, first washing step, first elution step) We prepared the NG2 clone (BioLegend), an anti-Ng antibody (IgG1) whose epitope is residues 52-63 of human neurogranin (Ng). 100 μg of anti-Ng antibody was reacted with 4.95 mg of magnetic beads (Dynabeads M-270 Epoxy) in immobilization buffer (0.1 M phosphate buffer containing 1.5 M ammonium sulfate; pH 7.4) at 37°C for 16 to 24 hours to produce antibody beads.

[0087] Stable isotope-labeled Ng50-78 (SIL-Ng50-78) was spiked into human plasma at 300 pM. 250 μL of the plasma was mixed with 250 μL of binding buffer (surfactants [0.2% DDM and 0.2% NTM], 800 mM GlcNAc, 100 mM Tris-HCl, 300 mM NaCl; pH 7.4) and incubated on ice for 5–60 min. The plasma was then mixed with antibody beads and shaken on ice for 1 h. The antibody beads were then washed three times with 100 μL of first wash buffer (surfactants [0.1% DDM and 0.1% NTM], 50 mM Tris-HCl, 150 mM NaCl; pH 7.4) and twice with 50 μL of 50 mM ammonium acetate buffer. The antibody beads were then contacted with a first acidic solution (50 mM glycine buffer containing a surfactant [0.1% DDM]; pH 2.8) to elute the Ng-related peptides into the first acidic solution, thereby obtaining a first eluate containing the Ng-related peptides.

[0088] (Neutralization process) The first eluate was mixed with a neutral buffer (surfactant [0.2% DDM], 800 mM GlcNAc, 300 mM Tris-HCl, 300 mM NaCl; pH 7.4) to obtain a purified solution.

[0089] (Second binding step, second washing step, second elution step) The purified solution was mixed with the antibody beads and shaken on ice for 1 hour. The antibody beads were then washed five times with 50 μL of second wash buffer (surfactant [0.1% DDM], 150 mM Tris-HCl, 150 mM NaCl; pH 7.4), twice with 50 μL of 50 mM ammonium acetate buffer, and once with 30 μL of water. The antibody beads were then contacted with a second acidic solution (70% (v / v) acetonitrile aqueous solution containing 5 mM hydrochloric acid and 0.1 mM methionine; pH 2.3) to elute the Ng-related peptides into the second acidic solution. This yielded a second eluate containing Ng-related peptides.

[0090] (Analysis process) A MALDI-TOF MS system (Shimadzu Corporation) was used as the mass spectrometer. A 0.5 mg / mL CHCA / 0.2% (w / v) MDPNA matrix solution was prepared using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix for Linear TOF, methylenediphosphonic acid (MDPNA) as the matrix additive, and acetonitrile as the solvent. 0.5 μL of the matrix solution was dispensed into four wells of a MALDI plate (μFocus MALDI plate 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ)) and allowed to dry. The second eluate was dispensed into these four wells and placed in the wells.

[0091] Next, MALDI-TOF MS was run to detect Ng-related peptides. Mass spectral data were acquired using an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) in positive ion mode with Linear TOF. Each well contained 400 spots, with 16,000 shots accumulated. The m / z values of the Linear TOF data were expressed as the average mass of the peaks. The m / z values were calibrated using external standards: human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, bovine insulin, and cytochrome c.

[0092] <Examples 1 to 3> The analysis was carried out in the same manner as in Reference Example 1, except that the surfactants and their concentrations contained in each liquid were changed to those shown in Table 1.

[0093] <Comparative Examples 1 to 3> The analysis was carried out in the same manner as in Reference Example 1, except that the surfactants and their concentrations contained in each liquid were changed to those shown in Table 1.

[0094] [Table 1]

[0095] The abbreviations in the table, their carbon numbers and critical micelle numbers are shown below. TC12: Trehalose C12 DDM: n-Dodecyl-β-D-maltoside UDM: n-Undecyl-β-D-maltoside DM: n-Decyl-β-D-maltoside NTM:n-Nonyl-β-D-thiomaltoside OG: n-Octyl-β-D-glucoside

[0096] [Table 2]

[0097] <Analysis results> Figure 1 shows one mass spectrum (one well) obtained in Reference Example 1. Peaks corresponding to the masses of SIL-Ng50-78 and Ng43-75 were extracted from the detected peaks, and the S / N ratio of each peak displayed in AXIMA Performance analysis software (Shimadzu Biotach Launchpad: Shimadzu / KRATOS, Manchester, UK) was used as an index of sensitivity. The average value of the mass spectra from four wells was used as the S / N ratio. Table 3 shows the amino acid sequences and theoretical average m / z values of SIL-Ng50-78 and Ng43-75. Ng43-75 is an endogenous Ng peptide found in plasma.

[0098] [Table 3]

[0099] In each Example and Comparative Example, the S / N ratios at the peaks of SIL-Ng50-78 and Ng43-75 were calculated from the mass spectra of 4 wells in the same manner as in Reference Example 1.

[0100] Next, for each peak of SIL-Ng50-78 and Ng43-75, the relative ratio of the S / N of Examples 1 to 3 to the S / N of Reference Example 1, and the relative ratio of the S / N of Comparative Examples 1 to 3 to the S / N of Reference Example 1 were calculated.

[0101] A series of analytical experiments consisting of the above-mentioned Reference Example, Examples, and Comparative Examples were repeatedly carried out to obtain analytical data (the above-mentioned relative ratios) for 4 to 5 runs, and then the average value of these 4 to 5 runs was calculated. The results are shown in Figures 2 and 3.

[0102] As is clear from Figures 2 and 3, compared to Reference Example 1, Examples 1 to 3 showed an improvement in sensitivity (S / N) of 1.5 times or more, and Example 1 showed an improvement in sensitivity of 3 times or more. As a result, this analytical method can detect Ng fragment peptides from blood with high sensitivity, and can reliably measure Ng fragment peptides in blood. On the other hand, Comparative Examples 1 to 3 showed the same sensitivity as Reference Example 1.

Claims

1. A method for analyzing neurogranin-related peptides contained in blood, comprising: a binding step of contacting the blood with an antibody-immobilized support in a binding solution to obtain a conjugate in which the neurogranin-related peptide is bound to the antibody of the antibody-immobilized support; a washing step of washing the conjugate with a washing solution; an elution step of contacting the washed conjugate with an acidic solution to obtain an eluate in which the neurogranin-related peptide is eluted into the acidic solution; a detection step of detecting the neurogranin-related peptide in the eluate by mass spectrometry; In order, the binding solution and the washing solution both contain a surfactant; the number of carbon atoms in the hydrophobic group of the surfactant is 9 or more and 11 or less; Method for analyzing neurogranin-related peptides.

2. The analytical method according to claim 1 , wherein the acidic solution contains an organic solvent.

3. The analytical method according to claim 1 , wherein the mass spectrometry is matrix-assisted laser desorption / ionization.

4. By including a surfactant in the binding solution, the number of carbon atoms in the hydrophobic group of which is 9 or more and 11 or less, non-specific adsorption of components other than neurogranin-related peptides in the blood onto the antibody-immobilized carrier is suppressed, and The analytical method according to claim 1, wherein the washing solution contains a surfactant having a hydrophobic group with 9 to 11 carbon atoms, thereby removing highly hydrophobic components other than neurogranin-related peptides from the blood.

5. A method for analyzing neurogranin-related peptides contained in blood, comprising: a first binding step of contacting the blood with a first antibody-immobilized support in a binding solution to obtain a first conjugate in which the neurogranin-related peptide is bound to the antibody of the first antibody-immobilized support; a first washing step of washing the first bond with a first washing solution; a first elution step of contacting the washed first conjugate with a first acidic solution to obtain a first eluate in which the neurogranin-related peptide is eluted in the first acidic solution; a neutralization step of mixing the first eluate with a neutral buffer to obtain a purified solution; a second binding step of contacting the purified solution with a second antibody-immobilized support to obtain a second conjugate in which the neurogranin-related peptide is bound to the antibody of the second antibody-immobilized support; a second washing step of washing the second bonded body with a second washing solution; a second elution step of contacting the washed second conjugate with a second acidic solution to obtain a second eluate in which the neurogranin-related peptide is eluted in the second acidic solution; a detection step of detecting the neurogranin-related peptide in the second eluate by mass spectrometry; In order, the binding solution, the first washing solution, and the second washing solution all contain a surfactant; A method for analyzing a neurogranin-related peptide, wherein the number of carbon atoms in the hydrophobic group of the surfactant is 9 or more and 11 or less.

6. The analytical method according to claim 5 , wherein the second acidic solution contains an organic solvent.

7. the first acidic solution contains a surfactant; 6. The analytical method according to claim 5, wherein the number of carbon atoms in the hydrophobic group of the surfactant is 9 or more and 11 or less.

8. The analytical method according to claim 5 , wherein the mass spectrometry is matrix-assisted laser desorption / ionization.

9. By including a surfactant in the binding solution, the number of carbon atoms in the hydrophobic group of which is 9 or more and 11 or less, non-specific adsorption of components other than neurogranin-related peptides in the blood onto the first antibody-immobilized carrier is suppressed, and The analytical method described in claim 5, wherein the first washing solution and the second washing solution contain a surfactant having a hydrophobic group with 9 to 11 carbon atoms, thereby removing highly hydrophobic components other than neurogranin-related peptides from the blood.

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