Method for preparing sample solution containing neuropartin-related peptide and method for analyzing

Abstract

Provided are: a method for analyzing a neurogranulin-related peptide, which is capable of suppressing variations in analysis results; and a method for producing a biological sample containing a neurogranulin-related peptide, which is used in the method. A method for producing a sample solution containing a neurogranular protein-related peptide, in which a biological sample containing a neurogranular protein-related peptide and an organic solvent having a relative polarity of 0.200-0.700 are mixed to produce a sample solution in which the final concentration of the organic solvent is 5.0 (v / v)% or more.

Description

Technical Field

[0001] The present invention relates to a method for preparing a sample solution containing neurogranin-related peptides and a method for analyzing neurogranin-related peptides. Background Art

[0002] Alzheimer's disease is the main clinical entity of dementia. The number of patients suffering from it has gradually increased in recent years, and the study of this disease has become increasingly important. The onset of Alzheimer's disease is closely related to Aβ-related peptides such as amyloid β (Aβ) produced by the cleavage of amyloid precursor protein (APP). In addition, it has been reported that the specific Aβ-related peptide ratio detected by combining immunoprecipitation with mass spectrometry to detect multiple Aβ-related peptides in the blood can be effectively used as a blood biomarker for amyloid accumulation in the brain (Non-patent Documents 1-2, Patent Documents 1-3).

[0003] On the other hand, in order to monitor the progression of Alzheimer's disease, various biomarkers are needed, in addition to the biomarkers for reflecting the accumulation of amyloid substances, it is also necessary to reflect the biomarkers of each process such as Tau accumulation and neurodegeneration. Among them, neurogranin is one of the biomarkers of neurodegeneration, and it is reported that it is increased in the cerebrospinal fluid (CSF) of Alzheimer's patients (non-patent literature 3, non-patent literature 4). In addition, it is also reported that in the brain of Alzheimer's patients, the fragmentation of neurogranin is promoted, and its fragment peptides are present in the blood, and then there are also translated and modified neurogranins such as acetylation or glutathionylation (non-patent literature 5). Therefore, it is expected that the means of confirming neurodegeneration by detecting neurogranin-related peptides such as neurogranin or its fragment peptides by biological samples such as blood and CSF.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: WO2015 / 178398

[0007] Patent Document 2: WO2017 / 47529

[0008] Patent Document 3: Japanese Patent Application Publication No. 2017-20980

[0009] Non-patent literature

[0010] Materials 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 plasmabiomarker surrogating cerebral amyloid deposition.Proc Jpn Acad Ser B PhysBiol Sci.2014:90(9):353-364.

[0011] Materials 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,ArahataY,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.

[0012] Materials 3:Portelius E,Olsson B,Hoglund K,Cullen NC,Kvartsberg H,Andreasson U,Zetterberg H,Sandelius A,Shaw LM,Lee VMY,Irwin DJ,Grossman M,Weintraub D,Chen-Plotkin A,Wolk DA,McCluskey L,Elman L,McBride J,Toledo JB,Trojanowski JQ,Blennow K.:Cerebrospinal fluid neurogranin concentration inneurodegeneration:relationship to clinical phenotypes and neuropathology.ActaNeuropathol.2018:136(3):363-376.

[0013] Non-patent literature 4: Thorsell A, Bjerke M, Gobom J, Brunhage E, Vanmechelen E, Andreasen N, Hansson O, Minthon L, Zetterberg H, Blennow K.: Neurogranin incerebrospinal fluid as a marker of synaptic degeneration in Alzheimer's disease. Brain Res. 2010; 1362:13-22.

[0014] Non-patent document 5: Kvartsberg H, Lashley T, Murray CE, Brinkmalm G, Cullen NC, Hoglund K, Zetterberg H, Blennow K, Portelius E.: The intact postsynaptic protein neurogranin is reduced in brain tissue from patients with familial and sporadic Alzheimer's disease. Acta Neuropathol. 2019; 137(1): 89-102. Summary of the invention

[0015] Problem that the invention aims to solve

[0016] In addition, as a biological sample for analyzing neurogranin-related peptides, blood samples are preferred because they can be collected by routine examinations and are less invasive. However, since the amount of neurogranin-related peptides present in blood is very small, there is a disadvantage that the analysis result values ​​of the same blood sample may vary greatly depending on the analysis method.

[0017] An object of the present invention is to provide a method for analyzing neurogranin-related peptides that can suppress variations in analysis results, and a method for preparing a biological sample containing neurogranin-related peptides used in the method.

[0018] Solutions for solving problems

[0019] The first preparation method of the present invention relates to a method for preparing a biological sample containing a neurogranin-related peptide, wherein the biological sample containing the neurogranin-related peptide and an organic solvent having a relative polarity of greater than 0.200 and less than 0.700 are mixed to prepare a sample solution in which the final concentration of the organic solvent is greater than 5.0 (v / v)%.

[0020] The first mode of analysis method using the first mode of preparation method of the present invention relates to an analysis method for neurogranin-related peptides, which comprises: a preparation step of implementing the first mode of preparation method; and a measurement step of implementing mass spectrometry, liquid chromatography, immunoassay or surface plasmon resonance using the above-mentioned sample solution.

[0021] Effects of the Invention

[0022] According to the production method of the first aspect of the present invention, the degradation of neurogranin-related peptides contained in the biological sample can be suppressed. In addition, according to the analysis method of the first aspect of the present invention, the variation in the analysis results of neurogranin-related peptides can be suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The graph shows the mass spectra of sample solutions containing Ng-related peptides at various DMSO concentrations (final concentration 0 to 5%). The vertical axis represents relative intensity, and the horizontal axis represents m / z.

[0024] Figure 2 The graphs show the mass spectra of sample solutions containing Ng-related peptides at various DMSO concentrations (concentrations 0 to 30%).

[0025] Figure 3 This is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and the DMSO concentration (final concentration 0 to 5%). The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents the final DMSO concentration.

[0026] Figure 4 It is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and the DMSO concentration (final concentration 0 to 30%).

[0027] Figure 5 This is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the DMSO concentration (final concentration 0 to 5%). The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents the final DMSO concentration.

[0028] Figure 6It is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the DMSO concentration (final concentration 0 to 30%).

[0029] Figure 7 The figure shows the mass spectra of the sample solution containing the Ng-related peptide of the comparative example at different incubation times. The vertical axis represents the relative intensity, and the horizontal axis represents m / z.

[0030] Figure 8 This is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and the incubation time in a comparative example. The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents the room temperature incubation time.

[0031] Fig. 9 This is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and the incubation time in Comparative Example. The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents the incubation time at room temperature.

[0032] Fig.10 This is a graph showing the relationship between the ratio of SIL-Ng50-75 to SIL-Ng50-78 and various solvents. The vertical axis represents the ratio of SIL-Ng50-75 to SIL-Ng50-78, and the horizontal axis represents various solvents (final concentration 10%).

[0033] Fig.11 This is a graph showing the relationship between the ratio of rNg1-75 to rNg1-78 and various solvents. The vertical axis represents the ratio of rNg1-75 to rNg1-78, and the horizontal axis represents various solvents (final concentration 10%).

[0034] Fig.12 The graph shows the mass spectra of sample solutions containing Ng-related peptides at various ACN concentrations (final concentration 0 to 20%). The vertical axis represents relative intensity, and the horizontal axis represents m / z.

[0035] Fig.13 Show Fig.12 An enlarged view of the area around m / z 2150-2540.

[0036] Fig.14 Show Fig.12 An enlarged view of the area around m / z 4800-5000. DETAILED DESCRIPTION

[0037] 1. First Implementation Method

[0038] 1-1. Preparation method

[0039] The method for preparing a sample solution containing a neurogranin-related peptide according to the first embodiment mixes a biological sample and an organic solvent having a relative polarity of 0.200 or more and 0.700 or less.

[0040] It should be noted that "neurogranin-related peptide" (hereinafter referred to as "Ng-related peptide") includes neurogranin, translated and / or modified neurogranin, and fragment peptides thereof.

[0041] Biological samples are samples containing Ng-related peptides, and examples thereof include body fluids such as blood, cerebrospinal fluid, urine, body secretions, saliva, sputum, and feces. Blood includes whole blood, plasma, serum, and the like. Blood may be obtained by centrifuging whole blood collected from an individual, freezing it, and the like. Blood is preferably used. Blood is less invasive than cerebrospinal fluid, and is also a sample for screening in physical examinations, and is easily available.

[0042] The relative polarity of the organic solvent is 0.200 or more and 0.700 or less. The lower limit of the relative polarity is preferably 0.300 or more, more preferably 0.370 or more, and most preferably 0.400 or more. The upper limit is preferably 0.600 or less, more preferably 0.500 or less, and most preferably 0.450 or less. The relative polarity is disclosed in detail in, for example, "Solvents and Solvent Effects in Organic Chemistry", Christian Reichards, Wiley-VCH Publishers, 3rd ed., 2003.

[0043] As such organic solvents, for example, acetone (0.355), dimethylformamide (0.386), dimethyl sulfoxide (0.444), acetonitrile (0.460), 2-propanol (0.546), ethanol (0.654), 1-butanol (0.586), 2-butanol (0.506), etc. can be listed. The numbers in brackets represent the values ​​of relative polarity. These can be used alone or in combination of two or more. From the viewpoint of being able to further reliably inhibit the decomposition of Ng-related peptides, dimethyl sulfoxide (DMSO) is preferably listed. On the other hand, from the viewpoint of being able to further detect Ng-related peptides with high sensitivity in addition to inhibiting the decomposition of Ng-related peptides, acetonitrile (ACN; Acetonitrile) is preferably listed.

[0044] In the preparation process, the biological sample and the organic solvent are mixed in such a manner that the final concentration of the organic solvent is 5.0 (v / v)% or more relative to the sample solution containing the prepared Ng-related peptide. The lower limit of the final concentration is preferably 10.0 (v / v)% or more, and the upper limit is, for example, 30.0 (v / v)% or less, preferably 20.0 (v / v)% or less. By setting the concentration to above the above lower limit, the decomposition inhibition of the Ng-related peptide brought about by the mixed organic solvent can be effectively exerted. In addition, by setting the concentration to below the above upper limit, the decrease in the detection sensitivity of the Ng-related peptide caused by the excessive addition of the organic solvent can be suppressed. Furthermore, when acetonitrile is used, it is preferably based on the above range, and further preferably 15.0 (v / v)% or more and 20.0 (v / v)% or less. By setting these concentration ranges, the Ng-related peptide can be detected with higher sensitivity, so that, for example, in the mass spectrum obtained by the mass spectrometry method described later, more peaks of Ng-related peptides can be detected. The amount of the organic solvent added is, for example, 5.0 parts by volume or more, preferably 10.0 parts by volume or more, and for example, 200 parts by volume or less, preferably 50.0 parts by volume or less, relative to 100 parts by volume of the biological sample.

[0045] In the preparation process, in order to set the above-mentioned final concentration, it is preferred to add a buffer as needed. As the buffer, for example, Tris buffer, phosphate buffer, HEPES buffer, ammonium acetate buffer, etc. can be listed. Thus, the pH of the sample solution can be made neutral and reach the above-mentioned final concentration, and it is easy to purify in the purification process described later. The pH of the buffer is preferably neutral, for example, pH 6.0 or more, preferably 6.5 or more, and for example, 8.5 or less, preferably 8.0 or less. The mixing ratio of the buffer relative to the prepared sample solution containing the Ng-related peptide is, for example, 5.0 (v / v)% or more, preferably 10.0 (v / v)% or more, and for example, 80.0 (v / v)% or less, preferably 40.0 (v / v)% or less. In addition, relative to 100 parts by volume of the organic solvent, it is 50.0 parts by volume or more, preferably 100 parts by volume or more, and for example, 1000 parts by volume or less, preferably 500 parts by volume or less.

[0046] When the purification step described below is performed continuously, in the first binding step, a surfactant may be mixed with the biological sample together with the buffer solution described below, as in the binding solution described below.

[0047] Thus, a sample solution containing Ng-related peptides is prepared (manufactured). The sample solution contains a specific amount of a specific organic solvent, so that the decomposition of Ng-related peptides in the sample solution can be suppressed. That is, even if it is stored for a specified time, the decrease in the content of Ng-related peptides over time is suppressed, and the amount of Ng-related peptides is not easy to change after the biological sample is collected, and is almost constant. Therefore, even if the measurement is carried out after the specified time is stored (described later), the change in the detection concentration caused by the storage time and the deviation in the detection concentration can be suppressed. In addition, the decomposition occurring in the measurement process can also be suppressed. Therefore, the change in the detection concentration caused by the time of the measurement process can be suppressed and the deviation in the detection concentration can be suppressed.

[0048] The inventors of the present invention paid attention to Ng-related peptides contained in biological samples such as blood collected from organisms, and found that Ng-related peptides are decomposed (digested) over time in biological samples. Specifically, in Ng-related peptides, the C-terminus of the amino acid at position 75 of neurogranin is cleaved over time by the protease in the biological sample. That is, when Ng1-78 is cited as an example of an analysis object, it was found that when a blood sample is collected (separated) from a biological body and analyzed, Ng1-78 is gradually decomposed into Ng1-75 and Ng76-78 during the storage time from collection to analysis or the analysis preparation time, and Ng1-78 gradually changes (decreases) according to the time. Therefore, further in-depth research was conducted, and it was found that the decomposition of the amino acid can be inhibited by allowing a specific amount of a specific organic solvent to exist in the biological sample, thereby completing the preparation method of the present invention. It should be noted that Ng1-78 is often decomposed in the organism, but the organism will regenerate Ng1-78 accordingly, so Ng1-78 in the organism remains constant. The above-mentioned reduction begins when the organism is separated and made into a biological sample for analysis.

[0049] 1-2. Analysis process

[0050] The analysis method of the first embodiment is an analysis method for Ng-related peptides in a biological sample, which sequentially comprises a preparation process and a measurement process. The preparation process is the above-mentioned preparation method, and the measurement process implements mass spectrometry. As a preferred embodiment, a purification process is implemented in the measurement process. That is, the measurement process sequentially comprises: a purification process, purifying the Ng-related peptides in a sample solution containing the Ng-related peptides; and, a detection process, implementing mass spectrometry. This method is described in detail below.

[0051] [Purification process]

[0052] The purification process is preferably affinity purification. As affinity purification, it can be one purification or two purifications. From the viewpoint of detecting Ng-related peptides with higher sensitivity, it is preferred to implement two purifications. In this case, the purification process sequentially comprises the first binding process (an example of a binding process), the first cleaning process (an example of a cleaning process), the first elution process, a neutralization process, the second binding process, the second cleaning process and the second elution process (an example of an elution process). Each process is described in detail below.

[0053] (First bonding step)

[0054] In the first binding step, the sample solution containing the Ng-related peptide obtained in the preparation step is brought into contact with the first carrier, thereby binding the Ng-related peptide in the sample solution to the first carrier to obtain a first bound body.

[0055] The first carrier may be any carrier as long as it can bind to the Ng-related peptide, and examples thereof include antibody-immobilized carriers.

[0056] 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 having an antigen binding site capable of recognizing an Ng-related peptide or fragments thereof.

[0057] 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 chain, and H chain. More specifically, clones NG2, NG7, and EPR21152 and fragments thereof. The antibody may be either a monoclonal antibody or a polyclonal antibody.

[0058] Examples of the material of the first carrier include agarose, sepharose, dextran, silica gel, polyacrylamide, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, (meth)acrylic polymers, fluororesins, metal complex resins, glass, metals, and magnetic substances.

[0059] The shape of the first carrier may be any of spherical (including bead-like), plate-like, needle-like, irregular, etc., and may be a channel wall in a microdevice.

[0060] At this time, in addition to the first carrier, a binding solution may be further added as needed. The binding solution preferably contains a neutral buffer solution of a surfactant. As the buffer solution, for example, the same buffer solution as the above-mentioned buffer solution in the preparation process can be cited. The pH of the binding solution is, for example, pH 6.0 or more, preferably 6.5 or more, and, for example, 8.5 or less, preferably 8.0 or less.

[0061] As the surfactant contained in the binding solution, for example, a neutral surfactant having a hydrophobic group with a carbon number of 7 or more and 15 or less (preferably 9 or more and 11 or less) can be cited. This can suppress nonspecific adsorption to the first binding body or reduce ionization interference in mass spectrometry analysis. As such a surfactant, for example, surfactants having maltose in the hydrophilic part such as n-nonyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-decyl-β-D-maltoside, n-undecyl-β-D-maltoside (UDM: n-Undecyl-β-D-maltoside); surfactants having trehalose in the hydrophilic part such as α-D-pyranosyl α-D-pyranosyl glucoside monodecanoate (trehalose C10); surfactants having glucose in the hydrophilic part such as n-decyl-β-D-glucoside, etc. can be cited. These surfactants can be used alone or in combination of two or more.

[0062] The surfactant concentration in the binding solution is, for example, 0.01% (w / v) or more, preferably 0.05% (w / v) or more, and, for example, 10% (w / v) or less, preferably 3% (w / v) or less. When the surfactant concentration is within the above range, micelles are sufficiently formed, so the effect of the surfactant can be reliably exerted.

[0063] Before the first binding step, a pretreatment to remove antibodies such as IgG and IgM may be performed as necessary.

[0064] (1st cleaning step)

[0065] In the first cleaning step, after the first bonding step, the first bonded body is cleaned using a first cleaning solution.

[0066] The first cleaning solution is preferably a neutral buffer solution containing a surfactant. Thus, it is possible to effectively remove unnecessary components with high hydrophobicity (proteins, lipids, glycolipids, etc. in the blood). As the neutral buffer solution and surfactant in the first cleaning solution, the same neutral buffer solution and surfactant as those exemplified in the binding solution can be cited.

[0067] The surfactant concentration in the first cleaning solution is, for example, 0.01% (w / v) or more, preferably 0.02% (w / v) or more, and, for example, 5% (w / v) or less, preferably 2% (w / v) or less. When the surfactant concentration is within the above range, micelles are fully formed, so the effect of the surfactant can be reliably exerted.

[0068] The washing method may be a known method, and it is preferred to perform washing multiple times, for example, washing with a neutral buffer containing a surfactant, and then washing with a neutral buffer not containing a surfactant.

[0069] The neutral buffer solution not containing a surfactant may be the same neutral buffer solution as that exemplified in the binding solution. This can suppress the foaming caused by the surfactant remaining in the first binding body.

[0070] As the washing method, a conventional method can be used, and examples thereof include 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 can be further performed as necessary.

[0071] (First elution step)

[0072] In the first elution step, after the first washing step, the first binding body is brought into contact with the first acidic solution. As a result, the Ng-related peptide is dissociated from the first binding body and eluted into the first acidic solution. As a result, a first eluate containing the Ng-related peptide is obtained.

[0073] The first acidic solution may be 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.

[0074] The first acidic solution preferably contains a surfactant. This allows the Ng-related peptide to be more reliably dissociated from the first conjugate. In addition, the eluted Ng-related peptide is inhibited from adhering to containers such as test tubes and microplates. Therefore, the recovery rate of the Ng-related peptide can be reliably improved and the detection sensitivity can be improved. As the surfactant used in the first acidic solution, the same surfactant as the surfactant exemplified in the binding solution can be listed. In addition, the surfactant concentration in the first acidic solution is the same as the surfactant concentration in the first cleaning solution.

[0075] (Neutralization process)

[0076] In the neutralization step, after the first elution step, the first eluate is mixed with a neutral buffer solution, thereby neutralizing the first eluate to obtain a purified solution containing the Ng-related peptide.

[0077] The neutral buffer used in the neutralization process preferably contains a surfactant. Thus, nonspecific adsorption to the second binding body in the second binding process can be suppressed. As the neutral buffer and surfactant used in the neutralization process, the same neutral buffer and surfactant as those exemplified in the binding solution can be cited. In addition, the surfactant concentration in the neutral buffer is the same as the surfactant concentration in the first binding solution.

[0078] The pH of the obtained purified solution is neutral, for example, pH 6.0 or higher, preferably pH 6.5 or higher, and for example, pH 8.5 or lower, preferably pH 8.0 or lower. Thus, in the second binding step, the binding efficiency can be improved.

[0079] (Second bonding step)

[0080] In the second binding step, after the neutralization step, the purified solution is brought into contact with the second carrier, thereby binding the Ng-related peptide in the purified solution to the second carrier to obtain a second bound body.

[0081] The second carrier is preferably an antibody-immobilized carrier, and specific examples thereof include the same carriers as exemplified for the antibody-immobilized carriers in the first carrier.

[0082] (Second cleaning step)

[0083] In the second cleaning step, after the second bonding step, the second bonded body is cleaned using a second cleaning solution.

[0084] The second cleaning solution is preferably a neutral buffer solution containing a surfactant. Thus, it is possible to effectively remove, for example, highly hydrophobic unnecessary components (proteins, lipids, glycolipids, etc. in the blood). As the neutral buffer solution and surfactant used in the second cleaning solution, the same neutral buffer solution and surfactant as those exemplified in the binding solution can be cited. In addition, the surfactant concentration in the second cleaning solution is the same as the surfactant concentration in the first cleaning solution.

[0085] A known method can be adopted as the cleaning method. Specifically, the same method as the cleaning method exemplified in the first cleaning step can be implemented.

[0086] (Second elution step)

[0087] In the second elution step, after the second washing step, the second conjugate is brought into contact with the second acidic solution. Thus, the Ng-related peptide is dissociated from the second conjugate and eluted into the second acidic solution. As a result, a second eluate containing the Ng-related peptide is obtained.

[0088] Examples of the acidic aqueous solution constituting the second acidic solution include the same acidic solutions as those exemplified for the first acidic solution in the first elution step, and preferably hydrochloric acid is used.

[0089] The second acidic solution preferably contains a volatile organic solvent, thereby enabling the Ng-related peptide to be efficiently dissociated from the second binding body and eluted into the second acidic solution, thereby improving the recovery rate of the Ng-related peptide.

[0090] Examples of the volatile organic solvent include organic solvents that are miscible with water in any proportion, such as acetonitrile, methanol, ethanol, acetone, toluene, isopropanol, hexane, butanol, cyclohexane, ethylene glycol, benzene, chloroform, acetaldehyde, triethylamine, phenol, naphthalene, formaldehyde, tetrahydrofuran, ethyl acetate, etc., preferably acetonitrile, methanol, ethanol, acetone, isopropanol, etc. These organic solvents may be used alone or in combination of two or more.

[0091] 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 is, for example, 90% (v / v) or less, preferably 80% (v / v) or less. When the concentration is within the above range, the Ng-related peptide can be efficiently dissociated from the second carrier, and the sensitivity (S / N ratio) during mass spectrometry analysis can be improved.

[0092] The second acidic solution preferably further contains amino acids such as methionine. Thus, during the period from the time of being placed in the mass spectrometer to the start of analysis, the oxidation of Ng-related peptides can be reduced, and the detection sensitivity can be improved. 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.

[0093] The second acidic solution preferably also contains proteins above 9 kDa. Since the above-mentioned proteins are contained in the mass spectrometry analysis sample, the proteins act as proton acceptors during mass spectrometry analysis, and can efficiently ionize Ng-related peptides, resulting in greatly improved detection sensitivity of Ng-related peptides. The upper limit of the mass of the protein is, for example, below 100 kDa, preferably below 15 kDa. As such a protein, there is no limitation as long as the mass is above 9 kDa, and specifically, bovine serum albumin (BSA), cytochrome, ovalbumin, lysozyme, etc. can be cited. The concentration of the protein in the second acidic solution is, for example, above 10 nM, preferably above 30 nM, and, for example, below 600 nM, preferably below 150 nM.

[0094] [Inspection process]

[0095] Detection Step After the purification step, the second eluate is subjected to mass spectrometry to detect Ng-related peptides.

[0096] Ionization methods in mass spectrometry include, for example, MALDI (Matrix Assisted Laser Desorption / Ionization), ESI (Electrospray ionization), APCI (Atmospheric Pressure Chemical Ionization), etc. MALDI is preferably used from the viewpoint of being able to detect trace amounts of Ng-related peptides in a sample solution with high sensitivity.

[0097] Examples of mass spectrometry include, but are not limited to, TOF-MS (time-of-flight mass spectrometry), IT-MS (ion trap mass spectrometry), IT-TOF-MS (ion trap-time-of-flight mass spectrometry), FTICR-MS (Fourier transform ion cyclotron mass spectrometry), etc. When MALDI is used as an ionization method, these mass spectrometry methods are collectively referred to as MALDI-MS.

[0098] As a specific detection operation using MALDI-MS, for example, a solution containing a matrix is ​​first added to a MALDI plate and dried (crystallized) to thereby configure the matrix. Next, a second eluent is added to the matrix and dried to obtain a MALDI-MS sample. Next, the MALDI-MS sample is irradiated with a laser to ionize the Ng-related peptides, and the ionized peptides are detected using the detector of the above-mentioned device and output in the form of a mass spectrum. The mass spectrum is then analyzed to obtain analysis results such as the type and concentration of the Ng-related peptides contained in the biological sample.

[0099] Examples of the matrix include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, sinapinic acid, 3-aminoquinoline, etc. These matrices may be used alone or in combination of two or more.

[0100] As the solvent containing the matrix, for example, acetonitrile, trifluoroacetic acid, methanol, ethanol, water, etc. can be listed. These solvents can be used alone or in combination of two or more. The matrix concentration in the matrix-containing solution 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.

[0101] It is preferred to use a matrix additive in combination with the matrix. Examples of the matrix additive include compounds containing phosphonic acid groups, ammonium salts, and the like. From the viewpoint of being able to suppress the adverse effects of the residual cleaning solution on the background, compounds containing phosphonic acid groups are preferred. Examples of compounds containing phosphonic acid groups include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, methylenediphosphonic acid (MDPNA; Methylenediphosphonic acid), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, ethylenediaminotetraphosphonic acid, and the like. The concentration of the matrix additive in the matrix-containing solution 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.

[0102] According to this analysis method, it is possible to suppress the deviation of the analysis results of Ng-related peptides. In particular, when a biological sample collected from a living body is stored for a desired time, it is possible to suppress the deviation of the measured values ​​at each storage time (for example, the deviation of the peak intensity, ratio, concentration calculated from the peak intensity, etc. of various Ng-related peptides in the mass spectrum).

[0103] Based on the insights from the above-mentioned preparation method, the inventors conducted further in-depth research and found that when analyzing Ng-related peptides, by using a solution in which a specific amount of a specific organic solvent is pre-existing in a biological sample to inhibit the decomposition of Ng-related peptides as a measurement sample for analysis, it is possible to suppress the deviation of Ng peptide analysis caused by storage time or analysis preparation time, thereby completing the analysis method of the present invention.

[0104] 1-3. Modifications

[0105] In the above embodiment, mass spectrometry is performed as a detection process, but for example, liquid chromatography, immunoassay, or surface plasmon resonance may also be performed as a detection process. In these determination methods, except for using the sample solution containing Ng prepared in the above preparation process as a measurement sample, it can be implemented according to a known or conventional method. From the viewpoint that even trace amounts of Ng-related peptides in blood can be reliably detected, mass spectrometry is preferably performed as a detection process.

[0106] 2. Method

[0107] Those skilled in the art will appreciate that the above-mentioned multiple exemplary embodiments are specific examples of the following aspects.

[0108] (Item 1) A method for preparing a sample solution containing a neurogranin-related peptide according to one embodiment can prepare a sample solution in which a final concentration of the organic solvent is 5.0 (v / v) % or more by mixing a biological sample containing a neurogranin-related peptide with an organic solvent having a relative polarity of 0.200 or more and 0.700 or less.

[0109] (Item 2) The preparation method according to Item 1, wherein the final concentration of the organic solvent can be 5.0 (v / v) % or more and 20.0 (v / v) % or less.

[0110] (Item 3) The preparation method according to Item 1 or 2, wherein the biological sample may be blood.

[0111] (Item 4) The production method according to any one of Items 1 to 3, wherein a buffer solution may be further mixed into the biological sample.

[0112] (Item 5) The production method according to any one of Items 1 to 4, wherein the organic solvent may be dimethyl sulfoxide.

[0113] (Item 6) The production method according to any one of Items 1 to 4, wherein the organic solvent may be acetonitrile.

[0114] (Item 7) A method for analyzing neurogranin-related peptides according to one embodiment may comprise: a preparation step of implementing any one of the preparation methods 1 to 6; and a measurement step of implementing mass spectrometry, liquid chromatography, immunoassay or surface plasmon resonance using the sample solution.

[0115] (Item 8) The analysis method according to Item 7, wherein the measurement step may include: (a) a binding step, wherein the sample solution is contacted with a carrier, and the neurogranin-related peptide is bound to the carrier to obtain a conjugate; (b) a washing step, wherein the conjugate is washed with a washing solution; (c) an elution step, wherein the conjugate is contacted with an acidic solution, and the neurogranin-related peptide is eluted into the acidic solution to obtain an eluate; and (d) a detection step, wherein the neurogranin-related peptide in the eluate is detected by mass spectrometry.

[0116] (Item 9) The analysis method according to Item 8, wherein the ionization method of the mass spectrometry analysis method may be a matrix-assisted laser desorption ionization method.

[0117] Example

[0118] Next, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

[0119] <Example 1>

[0120] [Preparation process]

[0121] 50 μL of DMSO (dimethyl sulfoxide) was mixed into 200 μL of a buffer solution containing a surfactant (0.1% n-undecyl-β-D-maltoside (UDM), 800 mM GlcNAc, 100 mM Tris-HCl, 300 mM NaCl; pH 7.4) to prepare a buffer solution containing DMSO with a DMSO concentration of 20.0 (v / v)%. Next, the stable isotope-labeled Ng50-78 (SIL-Ng50-78; manufactured by TORAY RESEARCH CENTER, INC.) was mixed into the buffer solution to reach 50 pM and the recombinant Ng with a His tag (rNg; manufactured by abcam) was mixed into the buffer solution to reach 6 nM to prepare a buffer solution containing Ng·DMSO. It should be noted that the carbon atoms and nitrogen atoms of Pro and Val of SIL-Ng50-78 were respectively 13 C and 15 N substitution.

[0122] Next, 250 μL of a commercially available human plasma sample was mixed with 250 μL of a buffer solution containing Ng·DMSO to prepare a sample solution containing an Ng-related peptide having a final DMSO concentration of 10.0 (v / v) %. The sample solution containing the Ng-related peptide was incubated at room temperature for 3 hours.

[0123] [Purification process]

[0124] (First binding step, first washing step, first elution step)

[0125] The anti-Ng antibody (IgG1) clone NG2 (manufactured by BioLegend) with the 52nd to 63rd residue of human neurogranin (Ng) as the epitope was prepared. 5.5 mg of magnetic beads (Dynabeads M-270 Epoxy) and 100 μg of anti-Ng antibody were reacted in an immobilization buffer (0.1 M phosphate buffer containing 1.5 M ammonium sulfate; pH 7.4) at 37°C for 16 to 24 hours. Thus, antibody beads serving as an antibody immobilization carrier were prepared.

[0126] After the incubated sample solution containing Ng-related peptides is left to stand on ice for 5 to 60 minutes, the above-mentioned antibody beads are mixed and shaken on ice or at 4°C for 1 hour. The antibody beads are then washed 1 or 3 times with 100 μL of the first washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4) and 1 or 2 times with 50 μL of 50 mM ammonium acetate buffer. The antibody beads are then brought into contact with the first acidic solution (0.05% UDM, 50 mM glycine buffer; pH 2.8) to elute the Ng-related peptides into the first acidic solution. Thus, the first eluate containing the Ng-related peptides is obtained.

[0127] (Neutralization process)

[0128] The first eluate was mixed with a neutral buffer (0.1% UDM, 800 mM GlcNAc, 300 mM Tris-HCl, 300 mM NaCl; pH 7.4) to obtain a purified solution.

[0129] (Second Binding Step, Second Washing Step, Second Elution Step)

[0130] The purified solution was mixed with the antibody beads and shaken on ice or at 4°C for 1 hour. Thereafter, the antibody beads were washed 2 or 5 times with 50 μL of the second washing buffer (0.05% UDM, 50 mM Tris-HCl, 150 mM NaCl; pH 7.4), 1 or 2 times with 50 μL of 50 mM ammonium acetate buffer, and 1 time with 30 μL of water. Then, the antibody beads were contacted with the second acidic solution (70% (v / v) acetonitrile aqueous solution containing 5 mM hydrochloric acid and 0.1 mM methionine, 50 nM BSA), and the Ng-related peptides were eluted into the second acidic solution. Thus, the second eluate containing the Ng-related peptides was obtained.

[0131] [Inspection process]

[0132] As a mass spectrometer, AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) as a MALDI-TOF MS device was used. As a matrix for linear TOF, α-cyano-4-hydroxycinnamic acid (CHCA) was used, as a matrix additive, methylene diphosphonic acid (MDPNA) was used, and as a solvent, acetonitrile was used to prepare a 2 mg / mL CHCA / 0.2% (w / v) MDPNA matrix solution. 0.5 μL of the matrix solution was added to each of the 4 wells of the MALDI plate (μFocus MALDI plate 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ)), and after drying, 1 μL of the second eluent was added to each well and dried.

[0133] Next, MALDI-TOF MS was run to detect Ng-related peptides (SIL-Ng50-78, SIL-Ng50-75, rNg1-78 and rNg1-75). As a setting condition, mass spectral data was acquired by linear TOF in positive ion mode. For 1 well, 400 points were accumulated 16,000 times. The m / z value of linear TOF was expressed as the average mass of the peak. For m / z values, human angiotensin II and human ACTH fragment 18-39, bovine insulin oxide β chain, bovine insulin, and cytochrome c were used as external standards for calibration.

[0134] It should be noted that if Ng50-78 and Ng50-75 that are not labeled with stable isotopes, and non-recombinant Ng1-78 and Ng1-75 are used as the objects, they cannot be distinguished from Ng50-78, Ng50-75, Ng1-78 and Ng1-75 originally present in the plasma, and the degradation amount cannot be accurately evaluated. Therefore, Ng50-78 and Ng50-75 that are not present in human plasma and rNg1-78 and rNg1-75 with a His tag are used as the detection objects.

[0135] Table 1 shows the Ng-related peptides detected by mass spectrometry. The * in the table shows the theoretical average m / z of the divalent ion peak for rNg1-75 and rNg1-78.

[0136] [Table 1]

[0137]

[0138] [Change final concentration]

[0139] The DMSO concentration of the sample solution containing the Ng-related peptide obtained in the preparation step was changed to 0-25.0 (v / v%) (specifically, 0%, 0.05%, 0.25%, 0.5%, 2.5%, 5.0%, 10.0%, 15.0%, 20.0%, 25.0%) by changing the amount of DMSO and buffer. The preparation step to the detection step were carried out in the same manner as above.

[0140] These results are shown in Figure 1 to Figure 6 Specifically, the mass spectra of the sample solutions containing Ng-related peptides at various DMSO concentrations are shown in Figure 1 (final concentration 0-5%) and Figure 2 (final concentration 0 to 30%), and the relationship between the intensity ratio of the degraded peptide to the degraded peptide and the DMSO concentration is shown in the graph. Figures 3-4 (SIL-Ng50-75 / SIL-Ng50-78) and Figures 5-6 (rNg1-75 / rNg1-78).

[0141] <Comparative Example 1>

[0142] The preparation step to the detection step were carried out in the same manner as above except that DMSO was not mixed in the preparation step (ie, the DMSO concentration was set to 0%) and the incubation time was set to 0 minute, 1 hour, and 3 hours.

[0143] These results are shown in Figure 7 to Figure 9 Specifically, in order to verify the decomposition of Ng-related peptides collected from a living body, the mass spectra at various incubation times when a sample solution containing Ng-related peptides without DMSO was used are shown in FIG. Figure 7 The relationship between the intensity ratio of the degraded peptide to the degraded peptide and the incubation time is shown in the graph. Figure 8 (SIL-Ng50-75 / SIL-Ng50-78) and Fig. 9 (rNg1-75 / rNg1-78).

[0144] <Investigation 1>

[0145] (1) Decomposition of Ng in plasma without DMSO addition

[0146] according to Figure 7 Each time the incubation time increased, the peak intensity of SIL-Ng50-75 and the peak intensity of rNg1-75 increased. Figure 8 and Fig. 9The ratio of SIL-Ng50-75 to intact SIL-Ng50-78 and the ratio of rNg1-75 to intact rNg1-78 increased. This indicates that cleavage occurs at the C-terminal end of Ng75 over time under the influence of proteases in plasma.

[0147] (2) Inhibitory effect of Ng decomposition brought about by DMSO addition

[0148] Figure 1 to Figure 6 The results are shown in Table 1 after 3 hours of incubation in a state where the sample solution was supplemented with DMSO. Figure 1 and Figure 2 , the peaks of SIL-Ng50-75 and rNg1-75 are almost identical to the peaks of noise from the range of DMSO concentration of 5.0% or more. Figure 3 to Figure 6 The ratio of SIL-Ng50-75 to intact SIL-Ng50-78, and the ratio of rNg1-75 to intact rNg1-78 decrease as the DMSO concentration increases, and especially become sufficiently low values ​​when the DMSO concentration is above 5.0%. It can be seen that the Ng decomposition inhibitory effect is fully exerted when the DMSO concentration is above 5.0%. In addition, the amount of intact SIL-Ng50-78 and rNg1-78 remains stable (constant) over time, so it can also be seen that in the mass spectrometry analysis results, the deviation of these analysis results can be suppressed for the measurement samples obtained from the same biological sample.

[0149] It should be noted that according to Figure 6 When the DMSO concentration is 25.0% or more, the ratio of rNg1-75 to intact rNg1-78 tends to increase. This is presumably because the excessive addition of DMSO changes the three-dimensional structure of rNg1-78 and reduces the antigen-antibody reactivity, resulting in a decrease in the peak intensity of rNg1-78.

[0150] <Example 2>

[0151] A sample solution containing Ng-related peptide was prepared in the same manner as in Example 1 except that acetone, dimethylformamide, ACN (acetonitrile), 2-propanol, or ethanol was mixed instead of DMSO to a final concentration of 10.0%, and incubated at room temperature for 3 hours.

[0152] <Comparative Example 2>

[0153] A sample solution containing Ng-related peptide was prepared in the same manner as in Example 1 except that methanol or toluene was mixed instead of DMSO to a final concentration of 10.0%, and incubated at room temperature for 3 hours.

[0154] The curve showing the intensity ratio of the degraded peptide to the degraded peptide at this time is shown in FIG. Fig.10 (SIL-Ng50-75 / SIL-Ng50-78) and Fig.11 (rNg1-75 / rNg1-78) Table 2 shows the relative polarities of the organic solvents used in Examples and Comparative Examples.

[0155] [Table 2]

[0156] Organic solvents Relative polarity water 1 Methanol 0.762 Ethanol 0.654 2-Propanol 0.546 ACN 0.460 DMSO 0.444 dimethylformamide 0.386 acetone 0.355 Toluene 0.099

[0157] <Investigation 2>

[0158] according to Fig.10 and Fig.11 It can be seen that acetone, dimethylformamide, ACN, 2-propanol and ethanol have the same Ng decomposition inhibitory effect as DMSO. It should be noted that this is believed to be due to (1) non-polar molecules and Ng decomposing enzymes in plasma undergo hydrophobic interactions, which cut off the hydrophobic interactions that support the three-dimensional structure of the Ng decomposing enzyme and denature the Ng decomposing enzyme. On the other hand, (2) the polar groups of the organic solvents form hydrogen bonds with the polar groups of the Ng decomposing enzymes, thereby denaturing the Ng decomposing enzymes. According to the balance of polarity and non-polarity caused by these (1) and (2), organic solvents with a relative polarity within a certain range (above 0.200 and below 0.700) are particularly effective in denaturing the Ng decomposing enzyme and reducing the Ng decomposing effect. However, the present invention is not limited to these inferred mechanisms.

[0159] <Example 3>

[0160] A sample solution containing Ng-related peptide was prepared in the same manner as in Example 1 except that ACN was mixed instead of DMSO to a final concentration of 0 to 30.0 (v / v%) (specifically, 0%, 0.05%, 0.25%, 0.5%, 2.5%, 5.0%, 10.0%, 15.0%, 20.0%, 25.0%, 30.0%). Thereafter, these were incubated at room temperature for 3 hours. Next, the purification step and the detection step were carried out in the same manner as in Example 1.

[0161] The mass spectrometry results at the final concentration of 0 to 20% are shown in Figure 12 to Figure 14 The Ng-related peptides detected by mass spectrometry are shown in Table 3. The * in the table represents the theoretical average m / z of the divalent ion peak.

[0162] [Table 3]

[0163]

[0164] <Investigation 3>

[0165] according to Figure 12 to Figure 14 When the ACN concentration is 10.0-20.0%, the peaks of specific Ng peptides (Ng33-78, Ng45-68, Ng45-68, Ng48-78, Ng46-78, Ng45-78, Ng39-78, Ng33-78) are clearly observed, so the signal intensity increases and the sensitivity improves. Furthermore, when the ACN concentration is 15.0-20.0%, in addition to the above, two divalent ion peaks of Ng46-78 and Ng39-78 are observed, and the sensitivity is further improved. On the other hand, when the ACN concentration is below 5.0% and above 25.0%, the above peaks are not clearly observed. In addition, the above peaks are not clearly observed in the mass spectra of the analysis results of the above organic solvents (DMSO, acetone, dimethylformamide, ACN, 2-propanol, ethanol, methanol, toluene) other than ACN.

[0166] Regarding the increased sensitivity of Ng-related peptides brought about by the addition of ACN, it is believed that ACN has the effect of weakening the hydrophobic interaction between proteins and peptides, thereby making it easier for Ng-related peptides adsorbed by proteins in plasma to be released and antibodies to more easily approach epitopes, but the present invention is not limited to these speculated mechanisms.

Claims

1. A method for preparing a sample solution containing a neurogranin-related peptide, wherein: A biological sample containing a neurogranin-related peptide and an organic solvent having a relative polarity of 0.200 or more and 0.700 or less are mixed to prepare a sample solution having a final concentration of the organic solvent of 5.0 (v / v) % or more.

2. The preparation method according to claim 1, wherein The final concentration of the organic solvent is 5.0 (v / v) % or more and 20.0 (v / v) % or less.

3. The preparation method according to claim 1, wherein The biological sample is blood.

4. The preparation method according to claim 1, wherein A buffer is also mixed into the biological sample.

5. A method for analyzing neurogranin-related peptides, comprising: A preparation step, comprising carrying out the preparation method according to any one of claims 1 to 4; and In the measurement step, the sample solution is used to perform mass spectrometry, liquid chromatography, immunoassay or surface plasmon resonance.

6. The analysis method according to claim 5, wherein: The measuring process comprises: (a) a binding step of contacting the sample solution with a carrier to bind the neurogranin-related peptide to the carrier to obtain a conjugate; (b) a cleaning step of cleaning the combined body using a cleaning solution; (c) an elution step, contacting the conjugate with an acidic solution to elute the neurogranin-related peptide into the acidic solution to obtain an eluate; and (d) a detection step of detecting the neurogranin-related peptide in the eluate by mass spectrometry.

7. The analysis method according to claim 6, wherein: The ionization method of the mass spectrometry is a matrix-assisted laser desorption ionization method.

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