Detection of amyloid beta by mass spectrometry

Abstract

To provide a method for detecting or quantifying amyloid beta.SOLUTION: Provided is a method for determining the amount of amyloid beta 40 (Aβ40) in a sample, including: (a) pre-treating a surface of a device in contact with the sample with an agent that prevents amyloid beta fragments from sticking to the surface; (b) digesting amyloid beta in the sample to produce Aβ40 comprising the sequence GAIIGLMVGGVV (SEQ ID NO: 2); (c) purifying Aβ40; (d) ionizing Aβ 40 to produce a precursor ion; and (e) generating one or more fragment ions of Aβ40; and (f) determining the amount of ions from step (d) or (e) or both by mass spectrometry, wherein the amount of ions is related to the amount of Aβ40 in the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-references to related patent applications This application is in accordance with U.S. Provisional Patent Application No. 62 / 234,027, filed on September 28, 2015, and And claiming the interests of U.S. Provisional Patent Application No. 62 / 277,772 filed on January 12, 2016 Each of these is incorporated herein by reference in whole.

[0002] The present invention relates to the detection or quantification of amyloid beta. In certain embodiments, the present invention A method for detecting amyloid beta or its fragments by mass spectrometry. Regarding. [Background technology]

[0003] Alzheimer's disease is the most common form of dementia affecting the elderly population. Luzheimer's disease is characterized by a progressive decline in cognitive abilities, particularly memory and learning. One characteristic of this disease is that it is composed of amyloid-beta (Aβ or A-beta) peptides. Examples include senile plaques.

[0004] When predicting or diagnosing Alzheimer's disease using current clinical diagnostic methods, the accuracy and sensitivity are as follows: Low. Immunoassays are biomarkers that predict the progression of Alzheimer's disease. It is currently available to detect myloid beta. However, currently available immunoassay Inter-facility variability is a concern in the observation results obtained using the Issey method.

[0005] Accurate and highly sensitive assay methods are needed to detect amyloid beta. [Overview of the project]

[0006] In this specification, mass spectrometry, such as tandem mass spectrometry, is used to analyze samples. A method for detecting or determining the amount of amyloid beta (Aβ) in a substance is provided.

[0007] In certain embodiments, the method provided herein for determining the amount of amyloid beta The method involves (a) purifying amyloid beta in the sample; and (b) the amyloid beta in the sample. Ionizing myloid-beta; and (c) mass spectrometry, amyl This includes determining the amount of amyloid-beta ions, and the amount of amyloid-beta ions in the sample. It is related to the amount of amyloid-beta inside.

[0008] In certain embodiments, the method provided herein for determining the amount of amyloid beta The method involves (a) purifying amyloid beta in the sample; and (b) the amyloid beta in the sample. (c) Ionizing myloid-beta to produce amyloid-beta precursor ions. (d) generating ions of one or more amyloid-beta fragments; and (d) mass Spectrometry reveals the ions from step (c) or (d) or both. Including determining the quantity, the amount of amyloid-beta ions in the sample is the amount of amyloid-beta ions. It is related to the amount of ta.

[0009] In certain embodiments, the method provided herein for determining the amount of amyloid beta The method involves (a) digesting amyloid beta in a sample to obtain one or more amyloid beta (b) generating amyloid-beta fragments; (b) the one or more amyloid-beta fragments (c) Purifying the amyloid-beta fragments; (c) Ionizing the one or more amyloid-beta fragments to produce precursor ions; (d) generating one or more fragment ions ; and (e) determining, by mass spectrometry, the amount of ions from step (c) or (d) or both, where the amount of ions is related to the amount of amyloid beta fragments in the sample.

[0010] In certain embodiments, a method for determining the amount of amyloid beta 42 (Aβ42) is provided in this specification. In some embodiments, the Aβ42 fragment includes the sequence GAIIG LMVGGVVIA (SEQ ID NO: 4). In some embodiments, the method comprises: (a) digesting amyloid beta in a sample to produce amyloid beta 42 (Aβ42); (b) purifying Aβ42; (c) ionizing Aβ42 to produce precursor ions; (d) generating one or more fragment ions of Aβ42; and (e) determining, by mass spectrometry, the amount of ions from step (c) or (d) or both, where the amount of ions is related to the amount of Aβ42 in the sample.

[0011] In certain embodiments, a method for determining the amount of amyloid beta 40 (Aβ40) is provided in this specification. In some embodiments, the Aβ40 fragment includes the sequence GAIIG LMVGGVV (SEQ ID NO: 2). In some embodiments, the method comprises: (a) digesting amyloid beta in a sample to produce amyloid beta 40 (Aβ40); (b) purifying Aβ40; (c) ionizing Aβ40 to produce precursor ions; (d) generating one or more fragment ions of Aβ40; and (e ​​​​​​​​​)Determining the amount of ions from step (c) or (d) or both by mass spectrometry, wherein the amount of ions is related to the amount of Aβ40 in the sample. The amount of ions is related to the amount of Aβ40 in the sample.

[0012] In certain embodiments, methods for determining the amounts of amyloid beta 42 (Aβ42) and amyloid beta 40 ( Aβ40) are provided herein. In some embodiments, the method comprises: (a) digesting amyloid beta in a sample to generate amyloid beta 42 (Aβ4 2) and amyloid beta 40 (Aβ40); (b) purifying Aβ42 and Aβ4 0; (c) ionizing Aβ42 and Aβ40 to produce precursor ions ; (d) generating one or more fragment ions of Aβ42 and Aβ40 ; and (e) determining the amount of ions from step (c) or (d) or both by mass spectrometry, wherein the amount of ions is related to the amounts of Aβ42 and A β40 in the sample. The amount of ions is related to the amounts of Aβ42 and Aβ40 in the sample.

[0013] In certain embodiments, methods for determining the ratio of amyloid beta 42 (Aβ42) to amyloid beta 40 ( Aβ40) are provided herein. In some embodiments , the method comprises: (a) digesting amyloid beta in a sample to generate amyloid beta 42 (A β42) and amyloid beta 40 (Aβ40); (b) purifying Aβ42 and A β40; (c) ionizing Aβ42 and Aβ40 to produce precursor ions ; (d) generating one or more fragment ions of Aβ42 and Aβ40 ; and (e) determining the amount of ions from step (c) or (d) or both by mass spectrometry, wherein the amount of ions is related to the amounts of Aβ42 and Aβ40 in the sample. (f) Determine the amount of ions from both; and (f) Determine the ratio of Aβ42 to Aβ40. This includes determining the ratio of Aβ40 to Aβ42. In some embodiments, the method determines the ratio of Aβ40 to Aβ42. This includes making a decision.

[0014] In a particular embodiment, a method for diagnosing or predicting Alzheimer's disease or dementia, The amount of amyloid beta in the test sample is determined by mass spectrometry. If amyloid-beta levels are abnormal, including [specifically, amyloid-beta], Alzheimer's disease may be predicted or diagnosed. A method is provided herein that allows for the following: In some embodiments, the method involves (a) sample (b) Purifying the amyloid beta in the sample; (b) Purifying the amyloid beta in the sample (c) to ionize; and the amount of amyloid-beta ions by mass spectrometry (d) the amount of amyloid beta ions in the sample This may include associating it with the amount of amyloid beta, and if the level of amyloid beta is abnormal, Luzheimer's disease is predicted or diagnosed. In some embodiments, the method involves amyloid This includes determining the ratio of the amide fragments. In some embodiments, the method involves determining the ratio of the amide fragments. Determining the ratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40). This includes the following. In some embodiments, the method involves the treatment of amyloid beta 40 (Aβ42) This includes determining the ratio of amyloid-beta-42 (Aβ40).

[0015] In certain embodiments, the method provided herein involves the surface of an instrument that comes into contact with a sample. This includes pretreatment. In some embodiments, pretreatment involves applying an amylose coating to the surface of the equipment. Pre-coating with an agent that prevents idobateta or its fragments from adhering to the surface. This includes the following. In some embodiments, the pretreatment includes pretreatment with a lysate. In some embodiments, the pretreatment includes pretreatment with an E. coli lysate. In some embodiments, the E. coli lysate includes trypsin-digested E. coli lysate. In that embodiment, the pre-treated equipment includes test tubes or plates, pipette tips, Examples include sample preparation equipment, liquid chromatography equipment, and mass spectrometry equipment. These are possible, but are not limited to these.

[0016] In certain embodiments, the methods provided herein involve amyloid beta or its flag. Treat the sample with a ment stabilizing agent, or incubate it together with the agent. This includes doing so. In some embodiments, the methods provided herein involve taking a sample This includes treatment with a myloid-beta antibody, or incubation with it. In some embodiments, the method provided herein involves a sample being divided into at least two different This includes treating with or incubating with an amyloid-beta antibody. In some embodiments, the amyloid-beta antibody binds to the C-terminus of amyloid-beta. Contains an antibody. In some embodiments, the amyloid-beta antibody is N- It contains an antibody that binds to the end. In some embodiments, it stabilizes amyloid beta. The drug contains an apolipoprotein. In some embodiments, it stabilizes amyloid beta. The agent used to induce the transformation includes apolipoprotein E2. In some embodiments, amyloid The agent that stabilizes beta includes apolipoprotein E4. In some embodiments, Drugs that stabilize amyloid beta include antibodies that bind to the C-terminus of amyloid beta. Antibodies that bind to the N-terminus of amyloid beta, apolipoprotein E2, apolipoprotein Includes quality E4, or a combination thereof. In some embodiments, the amylos provided herein The drug used to stabilize idobate provides stability at -70°C for at least one month. In some embodiments, the amyloid-beta stabilizing agents provided herein are , provides stability at -70°C for at least two months. In some embodiments, this specification The amyloid beta stabilizing agent provided in this book requires at least 3 months at -70°C. , providing stability. In some embodiments, amyloid beta provided herein The stabilizing agents impart stability over the freeze-thaw cycle. In the application form, the amyloid-beta stabilizing agent provided herein is at least Provides stability over two freeze-thaw cycles. In some embodiments, The amyloid-beta stabilizing agents provided in the document require at least three freeze-thaw cycles. Provides stability over the cycle. In some embodiments, provided herein Drugs that stabilize amyloid beta should be administered over at least four freeze-thaw cycles. , providing stability.

[0017] In certain embodiments, the method provided herein involves the extraction of amyloid beta in a sample. This includes digestion. In some embodiments, the methods provided herein involve enzymes This includes digesting amyloid beta. In some embodiments, the enzyme is Lys- C. In some embodiments, the method provided herein involves amyloyd by urea. This includes digesting dobeta. In some embodiments, urea is suitable for protein digestion. It has a concentration such that. In some embodiments, the urea is 6M urea. In this context, the method provided herein eliminates amyloid beta with urea and Lys-C. This includes the process of digestion. In some embodiments, digestion shortens the digestion time or improves digestive efficiency. This includes digesting under conditions that enhance the effect. In some embodiments, digestion is carried out in microwaves. This includes digestion. In some embodiments, the method provided herein is digested This includes determining the amount of amyloid beta.

[0018] In certain embodiments, the methods provided herein include extraction. The methods provided herein include mixed-mode anion exchange extraction. In terms of application methods, the methods provided herein include solid-phase extraction.

[0019] In certain embodiments, the method provided herein uses heated nitrogen to sample This includes eluting and drying the sample. In some embodiments, the sample is used for reconstruction. It is resuspended in the buffer.

[0020] In certain embodiments, sample purification includes liquid chromatography. In some embodiments, reversed-phase liquid chromatography is used as the liquid chromatography ( RPLC, high-performance liquid chromatography (HPLC), and high-turbulence liquid chromatography Examples include, but are not limited to, FI8 (HTLC). In a preferred embodiment... Liquid chromatography includes HPLC. In some embodiments, HPLC Lamb uses a medium (i.e., packing material) that facilitates the separation (i.e., fractionation) of chemical parts. Generally includes. Suitable columns include C-4, C-8, C-12, or C-18 columns. In a preferred embodiment, the suitable HPLC column is a C-4 column.

[0021] In certain embodiments, the method provided herein involves the application of amyloid beta to the device surface. This includes using equipment to reduce adhesion. In some embodiments, the equipment is PEE The device is equipped with a K (polyether ether ketone) tube or apparatus. In some embodiments, it is equipped with a K (polyether ether ketone) tube or apparatus. The equipment includes metal tubes or devices.

[0022] In certain embodiments, the methods provided herein include tandem mass spectrometry. In some embodiments, the method provided herein involves ionizing in positive mode. This includes, in some embodiments, the methods provided herein in negative mode This includes ionization. In some embodiments, the methods provided herein involve heating This includes ionization using the electrospray ionization method (HESI). In some embodiments, the method provided herein is electrospray ionization (E This includes ionization using SI. In some embodiments, as provided herein The method involves ionization using atmospheric pressure chemical ionization (APCI). In a particular embodiment, the method provided herein involves heated electrospray ionization. This involves using the HESI method to ionize in positive mode. In the implementation configuration, the collision energy is 5V to 60V. In some embodiments, the collision energy The energy is 10V to 50V. In some embodiments, the collision energy is 20 The voltage is V to 50V. In some embodiments, the collision energy is 20V to 45V. .

[0023] In certain embodiments, the method provided herein involves amyloid beta 40 (Aβ40) This includes detecting or determining the amount of ). In some embodiments, Aβ40 is sequence DA EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV(distribution Includes column number 1). In some embodiments, the method provided herein is for Aβ40 This includes detecting or determining the amount of fragments. In some embodiments, Aβ40 fragments The fragment contains the sequence GAIIGLMVGGVV (sequence number 2). Several implementations In this state, the Aβ40 fragment contains an N-terminal or C-terminal winged peptide. It includes the sequence. In some embodiments, the Aβ40 fragment is sequence number 2, and N It contains terminal or C-terminal flying peptides. In some embodiments, the flying peptide is a parent It is aqueous. In some embodiments, the flying peptide contains at least one amino acid Includes. In some embodiments, the flying peptide contains at least two amino acids. In some embodiments, the flying peptide contains at least three amino acids. In one embodiment, the flying peptide contains at least four amino acids. In the embodiment, the flying peptide contains at least 5 amino acids. Several embodiments The flying peptide then contains at least six amino acids. In some embodiments, The amount of Aβ40 fragment correlates with the amount of Aβ40 in the sample.

[0024] In certain embodiments, the methods provided herein involve amyloid beta 42 (Aβ42 This includes detecting or determining the amount of ). In some embodiments, Aβ42 is sequence DA EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (Sequence ID 3) is included. In some embodiments, the method provided herein is Aβ4 This includes detecting or determining the amount of fragment 2. In some embodiments, Aβ4 2 fragments contain the sequence GAIIGLMVGGVVIA (sequence number 4). In this embodiment, the Aβ42 fragment contains an N-terminal or C-terminal flying peptide. Includes sequence. In some embodiments, the Aβ42 fragment is sequence number 4, and the N-terminal It contains terminal or C-terminal flying peptides. In some embodiments, the flying peptide is hydrophilic. It is a type of avian. In some embodiments, the flying peptide contains at least one amino acid. In some embodiments, the flying peptide contains at least two amino acids. In some embodiments, the flying peptide contains at least three amino acids. In some embodiments, the flying peptide comprises at least four amino acids. In terms of form, the flying peptide contains at least five amino acids. In some embodiments The flying peptide contains at least 6 amino acids. In some embodiments, A The amount of β42 fragment correlates with the amount of Aβ42 in the sample.

[0025] In certain embodiments, the method provided herein involves the ratio of Aβ40 to Aβ42 ( This includes detecting or determining Aβ40:Aβ42). In some embodiments, this specification The method provided in this book is the ratio of Aβ40 fragment to Aβ42 fragment (Aβ This includes detecting or determining several fragments (Aβ42 fragment). In embodiments, the method provided herein relates to the ratio of Aβ42 to Aβ40 (Aβ42 This includes detecting or determining Aβ40. In some embodiments, provided herein The method used is the ratio of Aβ42 fragments to Aβ40 fragments (Aβ42 fragments This includes detecting or determining the Aβ40 fragment.

[0026] In certain embodiments, the ratio of Aβ42 to Aβ40, or the ratio of Aβ40 fragments to Aβ40 fragments. If the ratio of Aβ42 fragments is 0.6 or less, Alzheimer's disease is predicted. In some embodiments, Aβ42 relative to Aβ40 The ratio, or the ratio of Aβ42 fragments to Aβ40 fragments, is 0.5 or less. In that case, Alzheimer's disease is predicted or diagnosed. Several embodiments So, what is the ratio of Aβ42 to Aβ40, or the ratio of Aβ42 fragments to Aβ40 fragments? If the ratio of the genotype is 0.45 or less, Alzheimer's disease is predicted, or This is diagnosed. In some embodiments, the ratio of Aβ42 to Aβ40, or Aβ4 If the ratio of Aβ42 fragments to 0 fragments is 0.4 or less, Zheimer's disease is predicted or diagnosed. In some embodiments, Aβ40 The ratio of Aβ42 to Aβ40 fragments, or the ratio of Aβ42 fragments to Aβ40 fragments, A score of 0.35 or less indicates a predicted or diagnosed case of Alzheimer's disease. In some embodiments, the ratio of Aβ42 to Aβ40, or the Aβ40 fragment, is used. If the ratio of Aβ42 fragment to β is 0.3 or less, then Alzheimer's disease is Predicted or diagnosed as such. In some embodiments, Aβ40 versus Aβ4 The ratio of 2, or the ratio of Aβ42 fragment to Aβ40 fragment is 0.25 or so If the value is less than this, Alzheimer's disease is predicted or diagnosed. In terms of application methods, the ratio of Aβ42 to Aβ40, or the ratio of Aβ40 fragments to Aβ4 If the ratio of two fragments is 0.2 or less, Alzheimer's disease is predicted, It is diagnosed as such. In some embodiments, the ratio of Aβ42 to Aβ40, or A If the ratio of Aβ42 fragment to β40 fragment is 0.15 or less Alzheimer's disease is suspected or diagnosed.

[0027] In certain embodiments, the method involves one or more precursors of Aβ or its fragments. This includes generating ON. In some embodiments, at least one of the precursor ions is It has a mass / charge ratio of 1085.6 ± 0.5 or 1269.7 ± 0.5. In the embodiment, the method involves one or more Aβ or its fragments This may include generating ON. In some embodiments, a small amount of fragment ions One of them is 812.37±0.5, 869.4±0.5, 968.43±0.5, 86 9.39±0.5, 968.44±0.5, 1067.5±0.5, or 1180.57 It has a mass / charge ratio of ±0.5.

[0028] In certain embodiments, the methods provided herein include adding internal standards. In some embodiments, the internal standard includes an isotope-labeled internal standard. In the embodiment, the internal standard is: 13 C 15 Includes N label. In some embodiments, internal label Jun is, 13 C 15 It contains at least one Phe, Leu, or Met labeled with N. In some embodiments, at least one of the internal standard precursor ions is 1110.7±0 It has a mass / charge ratio of 0.5. In some embodiments, the method is one of the internal standards or This may include generating multiple fragment ions. In some embodiments, the fragment At least one of the ment ions is 768.48±0.5, 825.5±0.5, or 8 It has a mass / charge ratio of 82.52 ± 0.5.

[0029] In certain embodiments, the limit of quantification of the method is less than or equal to 10 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 5 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 4 ng / mL. In that embodiment, the limit of quantification of the method is less than or equal to 3 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 2 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 1 ng / mL. In the application method, the limit of quantification is less than or equal to 0.5 ng / mL. In the embodiments, the limit of quantification of the method is less than or equal to 0.2 ng / mL. In this embodiment, the limit of quantification of the method is less than or equal to 0.1 ng / mL.

[0030] In some embodiments, the detection limit of the method is less than or equal to 5 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 1 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.5 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.1 ng / mL. In some embodiments, the detection limit of the method is less than 0.05 ng / mL or equal to it. In some embodiments, the detection limit of the method is less than 0.01 ng / mL, or less than 0.01 ng / mL. It is equal to.

[0031] In some embodiments, amyloid beta is used as a derivative before mass spectrometry. It is not converted. In some embodiments, amyloid beta is converted by mass spectrometry. It is derivatized beforehand.

[0032] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is , cerebrospinal fluid (CSF). In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, The pull is either saliva or urine.

[0033] In some embodiments, the method involves using a sample in an amount sufficient to remove the protein, and the drug This may include adding the agent to the sample.

[0034] In this specification, unless otherwise specified, the singular forms "a," "an," and "the" are used. The word "1" implies a plural form. Therefore, for example, when you say "1 protein," it implies multiple proteins. Protein molecules are also included.

[0035] As used herein, the terms "purification" or "purification" mean removing a sample from a sample to obtain the desired result. Purification does not mean removing all substances other than the analyte being targeted. Rather, purification is about removing the target substance and Compared to other components in the sample, the detection of the analyte may interfere with the detection of the target analyte. This refers to a procedure for enriching the quantity of one or more analytes. The sample is as follows: This involves one or more interfering substances, for example, selected ami by mass spectrometry. This allows for the removal of one or more substances that interfere with the detection of parent and daughter ions of loid beta. It is refined by various means.

[0036] As used herein, the term "test sample" means any sample that may contain amyloid beta. This refers to a sample. As used herein, the term "body fluid" means an isolated sample from an individual's body. It means any possible fluid. For example, "body fluids" include blood, plasma, serum, bile, saliva, Examples include urine, tears, and sweat.

[0037] As used herein, the term "derivativeization" means reacting two molecules to create a novel It means forming a molecule. Derivatizing agents include isothiocyanate groups and dinitrofluorescein groups. Includes lophenyl groups, nitrophenoxycarbonyl groups, and / or phthalaldehyde groups, etc. obtain.

[0038] As used herein, the term "chromatography" refers to the process of transporting a substance by liquid or gas. When a chemical mixture flows around or above a stationary liquid or solid phase, the chemical entities This refers to the process of separating into components as a result of differential distribution.

[0039] As used herein, the terms "liquid chromatography" or "LC" refer to the process of creating fine particles. When a fluid permeates uniformly through a column made of a certain material, or through a capillary passage, This refers to a process in which one or more components of a fluid solution are selectively delayed. Alternatively, between multiple stationary phases and bulk liquid (i.e., mobile phase), this fluid is phase with respect to the stationary phase. This is caused by the distribution of components in a mixture during relative movement. Examples of "-" include reversed-phase liquid chromatography (RPLC) and high-performance liquid chromatography. Examples include HPLC (hydrochromatography) and highly turbulent liquid chromatography (HTLC).

[0040] As used herein, the terms "high-performance liquid chromatography" or "HPLC" mean The stationary phase, generally a densely packed column, applies force to the mobile phase under pressurized conditions. This refers to liquid chromatography, which offers a higher degree of separation.

[0041] As used herein, the terms "high turbulence liquid chromatography" or "HTLC" mean As a fundamental principle of separation, the turbulence of the substance being assayed through the column packing material is utilized. It refers to a form of chromatography that uses mass spectrometry. HTLC is a type of chromatography. Prior to analysis, it has been applied to prepare samples containing two unnamed drugs. Example For example, Zimmer et al., J. Chromatogr. A854: pp. 23-35 (199 See (9 years); U.S. Patent No. 5,968,367, Section 5 for further explanation of HTLCs. See also Nos. 919,368, 5,795,469, and 5,772,874. Those skilled in the art understand "turbulence." When a fluid flows slowly and smoothly, This type of flow is called "laminar flow." For example, flow moving at a low velocity through an HPLC column. The flow is laminar. In laminar flow, the movement of particles in the fluid is orderly, and the particles move in a straight line. This is the general practice. At higher speeds, the inertial force of water outweighs the frictional force of the fluid, resulting in disturbance. Flow occurs. Fluid that does not come into contact with irregular boundaries is slowed down by friction, or flows on an uneven surface. The fluid whose direction has changed due to the surface is "overtaken". When a fluid flows in a turbulent state, it swirls around. When the flow is swirling (or spiral), it becomes more "resistant" than when the flow is layered. There are many reference materials available that can help determine whether a fluid flow is laminar or turbulent. (For example, Turbulent Flow Analysis: Measurement) t and Prediction, PS Bernard & J.M.Wallace , John Wiley&Sons, Inc., (2000);An Introdu ction to Turbulent Flow Flow, Jean Mathie u&Julian Scott, Cambridge University Pres s (2001)).

[0042] As used herein, the terms "gas chromatography" or "GC" mean sample The mixture is vaporized, and a column containing a stationary phase consisting of liquid or particulate solids is used. It is injected into the flow of carrier gas (nitrogen or helium) moving through, and then the compound Based on its affinity for the stationary phase, chromatography is intended to separate the constituent compounds. It tastes good.

[0043] As used herein, the terms "large particle column" or "extraction column" refer to a column with particles of approximately 35 μm. This refers to a chromatography column containing an average particle size greater than m. Used in this context. In this case, the term "approximately" means ±10%. In a preferred embodiment, the column is straight It contains particles with a diameter of approximately 60 μm.

[0044] As used herein, the term "analytical column" refers to the substance in the sample that elutes from the column. Regarding this, sufficient separation is required to enable the determination of the presence or quantity of the analyte. This refers to a chromatography column with a chromatography plate. The substance is held in order to obtain a purified sample for further analysis. Many "extraction columns" have the general purpose of separating or extracting substances that are not otherwise extracted. In this case, a distinction is made. When used in this context, the term "approximately" means ±10%. In a preferred embodiment, the analytical column contains particles with a diameter of approximately 4 μm.

[0045] In this specification, for example, the terms "online automated method" or "online extraction" are used. The terms "online" or "inline," as used in this context, refer to situations where operator intervention is not required. This refers to procedures that are performed without requiring them. In contrast, the term "offline" as used herein When used in this context, it refers to a procedure that requires manual intervention by the operator. Therefore, sump If the sap is subjected to sedimentation and then the supernatant is manually loaded into an autosampler, The sedimentation and loading steps are offline from the subsequent steps. Various implementations of this method In terms of form, one or more steps may be performed using an online automated method.

[0046] As used herein, the terms "mass spectrometry" or "MS" mean the measurement of a compound. It refers to an analytical technique that identifies ions by their mass. MS (mass-mass analysis) identifies ions by their mass-to-charge ratio, or This refers to a method of filtering, detecting, and measuring based on "m / z". MS technology is (1) Ionizing a compound to form a charged compound; and (2) a charged compound This generally involves detecting the molecular weight of the compound and calculating the mass-to-charge ratio. It can be ionized and detected by any suitable means. "Mass spectrometer" is an ionizer. and generally include an ion detector. Generally, one or more target molecules are ionized. Then, the ions were introduced into a mass spectrographic apparatus, where magnetic and electric fields were used. The combination of these factors allows ions to follow paths in space that depend on their mass ("m") and charge ("z"). Follow this. For example, "Mass Spectrometry From Surface U.S. Patent No. 6,204,500, titled "Methods And Apparel The same year, titled "atus for Tandem Mass Spectrometry" No. 6,107,623, “DNA Diagnostics Based On Mass The same publication No. 6,268,144, titled "Surface Spectrometry" -Enhanced Photolabile Attachment And Rel ease For Desorption And Detection Of Ana Issues 6, 124, and 137 of the same publication, titled "lytes," by Wright et al., Prostate Cancer and Prostatic Diseases, Vol. 2: pp. 264-2676 Page (1999); and Merchant and Weinberger, Electro See Phoresis, Vol. 21: pp. 1164-1167 (2000).

[0047] As used herein, the term "operation in anion mode" means that anions are generated and detected. This refers to the mass spectrometry method used. The term "operation in cation mode" means... As used herein, this refers to a mass spectrometry method in which cations are generated and detected. do.

[0048] As used herein, the terms "ionization" or "the act of ionization" refer to one or more terms. A process that generates analyte ions with a net charge equal to the electron unit of the electron unit. Meaning: An anion is an ion that has a net negative charge of one or more electron units. On the other hand, a cation is an ion with a net positive charge of one or more electron units. It is.

[0049] As used herein, the terms "electron ionization method" or "EI method" refer to the gas phase [gaseous The method by which the target analyte in the [phase] or vapor phase interacts with the flow of electrons. This means that the electron impact on the analyte produces analyte ions, which are then measured by mass spectrometry. It can be a target of technology.

[0050] As used herein, the terms "chemical ionization method" or "CI method" refer to a method using a reagent gas (for example) For example, ammonia is subjected to an electron shock, and then there is an interaction between the reagent gas ions and the analyte molecules. This refers to the method by which analyte ions are formed.

[0051] As used herein, the terms "fast atomic bombing" or "FAB method" mean high energy A beam of atoms (often Xe or Ar) strikes a non-volatile sample, and the sample This refers to a method of desorption and ionization of molecules contained in a substance. The test sample is a viscous liquid. Body matrix, e.g., glycerol, thioglycerol, m-nitrobenzyl alcohol Lu, 18-crown-6-crown ether, 2-nitrophenyloctyl ether, S It dissolves in ruphorane, diethanolamine, and triethanolamine, etc. Compound or sa The selection of a suitable matrix for a sample is an experimental process.

[0052] As used herein, the term "matrix-assisted laser desorption / ionization" or "MA" "LDI" refers to various processes including photoionization, protonation, deprotonation, and cluster decay. Through a specific ionization pathway, the non-volatile sample desorbs and ionizes the analytes within the sample. This means a method of exposure to laser irradiation. In the case of MALDI, the sample is analyzed. It is mixed with an energy-absorbing matrix that promotes the desorption of molecules.

[0053] As used herein, the terms "surface-enhanced laser desorption / ionization method" or "SELDI method" are used. "This refers to various processes including photoionization, protonation, deprotonation, and cluster decay. The ionization pathway causes non-volatile samples to desorb and ionize the analytes within the sample. This refers to another method of exposure to laser irradiation. In SELDI, the sample is one or It generally binds to a surface that preferentially holds multiple target analytes, similar to MALDI. Furthermore, this process may also utilize energy-absorbing materials to promote ionization.

[0054] As used herein, the terms "electrospray ionization" or "ESI method" are defined as follows: The solution passes along a short capillary tube, and a positive or negative high pressure is applied to the end of the capillary tube. This refers to the method by which an electric potential is applied. When the solution reaches the end of the tube, it vaporizes (becomes atomized), In the solvent vapor, it forms a jet or spray consisting of very small droplets of the solution. The mist is placed in a slightly heated evaporation chamber to prevent condensation and evaporate the solvent. It flows. As the droplet becomes smaller, the surface charge density increases, and eventually, within the same charge, Naturally, repulsion occurs, triggering the release of ions and neutral molecules.

[0055] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI method" refer to ES It refers to a mass spectrometry method similar to I, however APCI is generated in the plasma at atmospheric pressure. Ions are produced through ion-molecular reactions. The plasma is used with a spray capillary and a counter electrode. It is maintained by the discharge during this time. The ions are then transported differentially by a series of skimmers. The stage is used to extract the dried and preheated N2 into the mass spectrometer. Using countercurrent gas flow can improve solvent removal. This is useful for analyzing less polar species. In such cases, gas-phase ionization in APCI may be more effective than ESI.

[0056] The terms "atmospheric pressure photoionization method" or "APPI method" as used herein refer to molecular The mechanism for photoionizing M is photon absorption and electron emission, which form the molecular ion M+. It refers to the form of the analytical method. Photon energy is generally just above the ionization potential. Molecular ions are not very easily dissociated. In many cases, chromatography is required. It is thought that samples can be analyzed without doing so, and therefore a considerable amount of time and expense can be saved. In the presence of water vapor or a protic solvent, the molecular ion removes H and produces MH+. This can be formed. This tends to occur when M has high proton affinity. Since the sum of M+ and MH+ is constant, this does not affect the accuracy of the quantitative analysis. Proton Drug compounds in polar solvents are usually observed as MH+, while nonpolar compounds, such as naphtham, are also observed. Talene or testosterone, etc., usually take the form of M+. Robb, DB, Cove y, TR and Bruins, AP (2000): For example, Robb et al., Atm ospheric pressure photoionization: An ion ization method for liquid chromatography - Mass spectrometry. Anal. Chem. Vol. 72 (No. 15): 3 See pages 653-3659.

[0057] As used herein, the terms "inductively coupled plasma method" or "ICP method" mean almost the same as the terms used herein. At a sufficiently high temperature such that the elements are atomized and ionized, the sample is partially ionized. This refers to the method of interacting with an turned-on gas.

[0058] As used herein, the term "field desorption method" means that a non-volatile test sample is ionized. This refers to a method in which a strong electric field is used to place the analyte ions on a surface and generate them. ru.

[0059] As used herein, the term "desorption" means the removal of an analyte from a surface, and / or This means introducing the analyte into the gas phase.

[0060] As used herein, the terms "limit of quantification" and "limit of quantification" are used. "Limit of Quantitation" or "LOQ" refers to the limit of quantitation, or the limit of quantity, of a measurement that has quantitative meaning. This refers to the point where it becomes clear. The response of the analyte in this LOQ is identifiable and individual. It is distinct and also has reproducibility with 20% precision and 80% to 120% accuracy.

[0061] As used herein, the terms “Limit of Detection” or “LOD” mean that a measured value is such that it is not related to the LOD. This is the point where the uncertainty becomes greater. The LOD is calculated from zero concentration to 2 standard deviations (SD). It is arbitrarily defined as follows.

[0062] As used herein, the “amount” of amyloid beta in a body fluid sample refers to the volume of the body fluid. This generally refers to the absolute value that reflects the mass of amyloid-beta detectable within the substance. However, the quantity This also considers the relative amount compared to the amount of other amyloid-beta. For example, body fluids The amount of amyloid beta inside is at the control level or normal level of normally present amyloid beta. It can be a quantity greater than or less than 1.

[0063] The term "approximately" is used herein in relation to quantitative measurements, excluding ion mass measurements. This means the displayed value is within ±10%. A mass spectrometry instrument measures the mass of a given analyte. When making a decision, it can hardly change. The term "approximately" refers to the mass of the ion or the mass of the ion. In the context of charge ratios, this means ±0.5 atomic mass units.

[0064] The above-described outline of the present invention is not limiting, and other characteristics and advantages of the present invention are as follows: This is evident from the modes for carrying out the invention described and from the claims. [Brief explanation of the drawing]

[0065] [Figure 1] This figure shows the sequences of Aβ40 (sequence number 1), Aβ40 fragment (sequence number 2), Aβ42 (sequence number 3), and Aβ42 fragment (sequence number 4). [Figure 2-1] This figure shows information on the liquid chromatography method. 0.1% FA dissolved in water was used as mobile phase A, and 0.1% FA dissolved in ACN was used as mobile phase B. [Figure 2-2] (Continued from Figure 2-1) This figure shows information on the liquid chromatography method. 0.1% FA dissolved in water was used as mobile phase A, and 0.1% FA dissolved in ACN was used as mobile phase B. [Figure 3] This figure shows the ion source conditions for heated electrospray ionization (HESI). [Figure 4] This figure shows the linear range of Aβ42 analysis. [Figure 5] This figure shows the linear range of Aβ40 analysis. [Figure 6-1] This figure shows examples of patient chromatograms for Aβ40, Aβ42, and an internal standard. [Figure 6-2] (Continued from Figure 6-1) This figure shows examples of patient chromatograms for Aβ40, Aβ42, and the internal standard. [Figure 7-1] This figure shows examples of patient chromatograms for Aβ40, Aβ42, and an internal standard. [Figure 7-2] (Continued from Figure 7-1) This figure shows examples of patient chromatograms for Aβ40, Aβ42, and the internal standard. [Figure 8] This figure shows the Aβ42:Aβ40 ratio for 211 subjects, including 91 patients with Alzheimer's disease, 66 patients with borderline Alzheimer's disease, and a control group with normal function. [Figure 9] This figure shows the Aβ42:Aβ40 ratio in female and male Alzheimer's disease patients. [Figure 10]This figure shows the Aβ42:Aβ40 ratio in groups stratified by age of Alzheimer's disease patients. [Figure 11] This figure shows a comparison of sensitivity between C8 and C4 analytical columns. [Figure 12] This figure shows the recovery rates of Aβ40 from treated and untreated tubes. [Figure 13] This figure shows the recovery rates of Aβ42 from treated and untreated tubes. [Figure 14] This figure shows patient Aβ values ​​(pg / mL) after sorting by Aβ40 reduction level. [Figure 15] This figure shows patient Aβ levels (pg / mL) after sorting by Aβ42 reduction level. [Figure 16] This figure shows the diagnosis of Alzheimer's disease in patients after sorting by Aβ42 / Aβ40 values. [Figure 17] This figure shows patient Alzheimer's disease diagnosis based on the Aβ42 / Aβ40 ratio. [Modes for carrying out the invention]

[0066] Mass spectrometry, such as tandem mass spectrometry, can be used to determine the amyloid molecules in a sample. Methods for detecting or determining the amount of β(Aβ) are provided herein. In certain embodiments The method provided herein for determining the amount of amyloid beta is (a) sample (b) Purifying amyloid beta in the sample (c) turning on; and (c) mass spectrometry reveals the presence of amyloid-beta ions. Including determining the quantity, the amount of amyloid-beta ions in the sample is the amount of amyloid-beta ions. It is related to the amount of ta.

[0067] In certain embodiments, the method provided herein for determining the amount of amyloid beta The method involves (a) purifying amyloid beta in the sample; and (b) the amyloid beta in the sample. (c) Ionizing myloid-beta to produce amyloid-beta precursor ions. (d) generating ions of one or more amyloid-beta fragments; and (d) mass Spectrometry reveals the ions from step (c) or (d) or both. Including determining the quantity, the amount of amyloid-beta ions in the sample is the amount of amyloid-beta ions. It is related to the amount of ta.

[0068] In certain embodiments, the method provided herein for determining the amount of amyloid beta The method involves (a) digesting amyloid beta in a sample to obtain one or more amyloid beta (b) generating amyloid-beta fragments; (b) the one or more amyloid-beta fragments (c) Purifying the amyloid beta; (c) Ionizing the amyloid beta in the sample (d) Production of amyloid-beta precursor ions; (d) One or more amyloid-beta fragments (e) generating ions; and (c) by mass spectrometry, step (c) This includes determining the amount of ions from (d) or both, and amyloid beta The amount of ON is related to the amount of amyloid beta in the sample.

[0069] In a particular embodiment, the method for determining the amount of amyloid beta 42 (Aβ42) is as described in the present invention. Provided in the details. In some embodiments, the Aβ42 fragment is sequence GAIIG Includes LMVGGVVIA (Sequence ID 4). In some embodiments, the method is (a) It digests amyloid beta in the sample to produce amyloid beta-42 (Aβ42). (b) Purify Aβ42; (c) Ionize Aβ42 to produce precursor ions (d) to produce one or more Aβ42 fragment ions; and (e) By mass spectrometry, from step (c) or (d) or both This involves determining the amount of ions, and the amount of ions is related to the amount of Aβ42 in the sample. ru.

[0070] In a particular embodiment, the method for determining the amount of amyloid beta 40 (Aβ40) is described in the present invention. Provided in the details. In some embodiments, the Aβ40 fragment is sequence GAIIG Includes LMVGGVV (Sequence ID 2). In some embodiments, the method is (a) sample The process involves digesting amyloid beta in the substance to produce amyloid beta-40 (Aβ40). (b) Purify Aβ40; (c) Ionize Aβ40 to produce a precursor ion. (d) generating one or more Aβ40 fragment ions; and (e ) Mass spectrometry is used to determine the results from step (c) or (d) or both. This involves determining the amount of ON, and the amount of ions is related to the amount of Aβ40 in the sample.

[0071] In certain embodiments, amyloid beta 42 (Aβ42) and amyloid beta 40 ( A method for determining the amount of Aβ40 is provided herein. In some embodiments, The method involves (a) digesting amyloid beta in the sample and amyloid beta-42 (Aβ4 2) and the production of amyloid beta 40 (Aβ40); (b) Aβ42 and Aβ4 (c) Purify 0; (c) Ionize Aβ42 and Aβ40 to produce precursor ions. (d) generating one or more Aβ42 and Aβ40 fragment ions. ; and (e) by mass spectrometry, step (c) or (d) or both This includes determining the amount of ions from one side, and the amount of ions is determined by the amount of Aβ42 and A in the sample. It is related to the amount of β40.

[0072] In a particular embodiment, amyloid beta 40 (Aβ40) A method for determining the ratio of 2(Aβ42) is provided herein. In some embodiments, The method is to (a) digest amyloid beta in the sample and amyloid beta-42(A (b) To produce β42) and amyloid beta 40 (Aβ40); (b) Aβ42 and A (c) Purify β40; (c) Ionize Aβ42 and Aβ40 to produce precursor ions. (d) To generate one or more Aβ42 and Aβ40 fragment ions. (e) by mass spectrometry, step (c) or (d) or (f) Determine the amount of ions from both; and (f) Determine the ratio of Aβ42 to Aβ40. This includes determining the ratio of Aβ40 to Aβ42. In some embodiments, the method determines the ratio of Aβ40 to Aβ42. This includes making a decision.

[0073] In a particular embodiment, a method for diagnosing or predicting Alzheimer's disease or dementia, The amount of amyloid beta in the test sample is determined by mass spectrometry. If amyloid-beta levels are abnormal, including [specifically, amyloid-beta], Alzheimer's disease may be predicted or diagnosed. A method is provided herein that allows for the following: In some embodiments, the method involves (a) sample (b) Purifying the amyloid beta in the sample; (b) Purifying the amyloid beta in the sample (c) to ionize; and the amount of amyloid-beta ions by mass spectrometry (d) the amount of amyloid beta ions in the sample This may include associating it with the amount of amyloid beta, and if the level of amyloid beta is abnormal, Luzheimer's disease is predicted or diagnosed. In some embodiments, the method involves amyloid This includes determining the ratio of the amide fragments. In some embodiments, the method involves determining the ratio of the amide fragments. Determining the ratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40). This includes the following. In some embodiments, the method involves the treatment of amyloid beta 40 (Aβ42) This includes determining the ratio of amyloid-beta-42 (Aβ40).

[0074] In certain embodiments, the method provided herein involves the surface of an instrument that comes into contact with a sample. This includes pretreatment. In some embodiments, pretreatment involves applying an amylose coating to the surface of the equipment. Pre-coating with an agent that prevents idobateta or its fragments from adhering to the surface. This includes the following. In some embodiments, the pretreatment includes pretreatment with a lysate. In some embodiments, the pretreatment includes pretreatment with E. coli lysate. In the embodiment, the E. coli lysate includes trypsin-digested E. coli lysate. Several embodiments In this state, pre-processed equipment includes test tubes or plates, pipette tips, and sample preparation. Examples include manufacturing apparatus, liquid chromatography apparatus, and mass spectrometry apparatus. However, this is not limited to these examples.

[0075] In certain embodiments, the methods provided herein involve amyloid beta or its flag. Treat the sample with a ment stabilizing agent, or incubate it together with the agent. This includes doing so. In some embodiments, the methods provided herein involve taking a sample This includes treatment with a myloid-beta antibody, or incubation with it. In some embodiments, the method provided herein involves a sample being divided into at least two different This includes treating with or incubating with an amyloid-beta antibody. In some embodiments, the amyloid-beta antibody binds to the C-terminus of amyloid-beta. Contains an antibody. In some embodiments, the amyloid-beta antibody is N- It contains an antibody that binds to the end. In some embodiments, it stabilizes amyloid beta. The drug contains an apolipoprotein. In some embodiments, it stabilizes amyloid beta. The agent used to induce the transformation includes apolipoprotein E2. In some embodiments, amyloid The agent that stabilizes beta includes apolipoprotein E4. In some embodiments, Drugs that stabilize amyloid beta include antibodies that bind to the C-terminus of amyloid beta. Antibodies that bind to the N-terminus of amyloid beta, apolipoprotein E2, apolipoprotein Includes quality E4, or a combination thereof. In some embodiments, the amylos provided herein The drug used to stabilize idobate provides stability at -70°C for at least one month. In some embodiments, the amyloid-beta stabilizing agents provided herein are , provides stability at -70°C for at least two months. In some embodiments, this specification The amyloid beta stabilizing agent provided in this book requires at least 3 months at -70°C. , providing stability. In some embodiments, amyloid beta provided herein The stabilizing agents impart stability over the freeze-thaw cycle. In the application form, the amyloid-beta stabilizing agent provided herein is at least Provides stability over two freeze-thaw cycles. In some embodiments, The amyloid-beta stabilizing agents provided in the document require at least three freeze-thaw cycles. Provides stability over the cycle. In some embodiments, provided herein Drugs that stabilize amyloid beta should be administered over at least four freeze-thaw cycles. , providing stability.

[0076] In certain embodiments, the method provided herein involves the extraction of amyloid beta in a sample. This includes digestion. In some embodiments, the methods provided herein involve enzymes This includes digesting amyloid beta. In some embodiments, the enzyme is Lys- C. In some embodiments, the method provided herein involves amyloyd by urea. This includes digesting dobeta. In some embodiments, urea is suitable for protein digestion. It has a concentration such that. In some embodiments, the urea is 6M urea. In this context, the method provided herein eliminates amyloid beta with urea and Lys-C. This includes the process of digestion. In some embodiments, digestion shortens the digestion time or improves digestive efficiency. This includes digesting under conditions that enhance the effect. In some embodiments, digestion is carried out in microwaves. This includes digestion.

[0077] In certain embodiments, the methods provided herein include extraction. The methods provided herein include mixed-mode anion exchange extraction. In terms of application methods, the methods provided herein include solid-phase extraction.

[0078] In certain embodiments, the method provided herein uses heated nitrogen to sample This includes eluting and drying the sample. In some embodiments, the sample is used for reconstruction. It is resuspended in the buffer.

[0079] In certain embodiments, sample purification includes liquid chromatography. In some embodiments, reversed-phase liquid chromatography is used as the liquid chromatography ( RPLC, high-performance liquid chromatography (HPLC), and high-turbulence liquid chromatography Examples include, but are not limited to, FI8 (HTLC). In a preferred embodiment... Liquid chromatography includes HPLC. In some embodiments, HPLC Lamb uses a medium (i.e., packing material) that facilitates the separation (i.e., fractionation) of chemical parts. Generally includes. Suitable columns include C-4, C-8, C-12, or C-18 columns. In a preferred embodiment, the suitable HPLC column is a C-4 column.

[0080] In certain embodiments, the method provided herein involves the application of amyloid beta to the device surface. This includes using equipment to reduce adhesion. In some embodiments, the equipment is PEE The device is equipped with a K (polyether ether ketone) tube or apparatus. In some embodiments, it is equipped with a K (polyether ether ketone) tube or apparatus. The equipment includes metal tubes or devices.

[0081] In certain embodiments, the methods provided herein include tandem mass spectrometry. In some embodiments, the method provided herein involves ionizing in positive mode. This includes, in some embodiments, the methods provided herein include negative mode This includes ionization. In some embodiments, the methods provided herein include adding This includes ionization using thermal electrospray ionization (HESI). In some embodiments, the methods provided herein are electrospray ionization methods. This includes ionization using (ESI). In some embodiments, as specified herein The method provided involves ionization using atmospheric pressure chemical ionization (APCI). Includes. In a preferred embodiment, the method provided herein is a heated electrospray. This includes ionization in positive mode using the ionization method (HESI). In some embodiments, the collision energy is 5V to 60V. The collision energy is 10V to 50V. In some embodiments, the collision energy The voltage is 20V to 50V. In some embodiments, the collision energy is 20V to 45V It is V.

[0082] In certain embodiments, the method provided herein involves amyloid beta 40 (Aβ40) This includes detecting or determining the amount of ). In some embodiments, Aβ40 is sequence DA EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV(distribution Includes column number 1). In some embodiments, the method provided herein is for Aβ40 This includes detecting or determining the amount of fragments. In some embodiments, Aβ40 fragments The fragment contains the sequence GAIIGLMVGGVV (sequence number 2). Several implementations In this state, the Aβ40 fragment contains a sequence that includes an N-terminal or C-terminal flying peptide. In some embodiments, the Aβ40 fragment is sequence number 2, and the N-terminus or C It contains terminally flying peptides. In some embodiments, the flying peptides are hydrophilic. In some embodiments, the flying peptide contains at least one amino acid. In some embodiments, the flying peptide contains at least two amino acids. In some embodiments, the flying peptide contains at least three amino acids. In this state, the flying peptide contains at least four amino acids. In some embodiments, The flight-forming peptide contains at least 5 amino acids. In some embodiments, the flight The peptide contains at least six amino acids. In some embodiments, Aβ40 The amount of lagment correlates with the amount of Aβ40 in the sample.

[0083] In certain embodiments, the methods provided herein involve amyloid beta 42 (Aβ42 This includes detecting or determining the amount of ). In some embodiments, Aβ42 is sequence DA EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (Sequence ID 3) is included. In some embodiments, the method provided herein is Aβ4 This includes detecting or determining the amount of fragment 2. In some embodiments, Aβ4 2 fragments contain the sequence GAIIGLMVGGVVIA (sequence number 4). In this embodiment, the Aβ42 fragment contains an N-terminal or C-terminal flying peptide. Includes sequence. In some embodiments, the Aβ42 fragment is sequence number 4, and the N-terminal It contains terminal or C-terminal flying peptides. In some embodiments, the flying peptide is hydrophilic. It is a type of avian. In some embodiments, the flying peptide contains at least one amino acid. In some embodiments, the flying peptide contains at least two amino acids. In some embodiments, the flying peptide contains at least three amino acids. In some embodiments, the flying peptide comprises at least four amino acids. In terms of form, the flying peptide contains at least five amino acids. In some embodiments The flying peptide contains at least 6 amino acids. In some embodiments, A The amount of β42 fragment correlates with the amount of Aβ42 in the sample.

[0084] In certain embodiments, the method provided herein involves the ratio of Aβ40 to Aβ42 ( This includes detecting or determining Aβ40:Aβ42). In some embodiments, this specification The method provided in this book is the ratio of Aβ40 fragment to Aβ42 fragment (Aβ This includes detecting or determining several fragments (Aβ42 fragment). In embodiments, the method provided herein relates to the ratio of Aβ42 to Aβ40 (Aβ42 This includes detecting or determining Aβ40. In some embodiments, provided herein The method used is the ratio of Aβ42 fragments to Aβ40 fragments (Aβ42 fragments This includes detecting or determining the Aβ40 fragment.

[0085] In certain embodiments, the ratio of Aβ42 to Aβ40, or the ratio of Aβ40 fragments to Aβ40 fragments. If the ratio of Aβ42 fragments is 0.6 or less, Alzheimer's disease is predicted. In some embodiments, Aβ42 relative to Aβ40 The ratio, or the ratio of Aβ42 fragments to Aβ40 fragments, is 0.5 or less. In that case, Alzheimer's disease is predicted or diagnosed. Several embodiments So, what is the ratio of Aβ42 to Aβ40, or the ratio of Aβ42 fragments to Aβ40 fragments? If the ratio of the genotype is 0.45 or less, Alzheimer's disease is predicted, or This is diagnosed. In some embodiments, the ratio of Aβ42 to Aβ40, or Aβ4 If the ratio of Aβ42 fragments to 0 fragments is 0.4 or less, Zheimer's disease is predicted or diagnosed. In some embodiments, Aβ40 The ratio of Aβ42 to Aβ40 fragments, or the ratio of Aβ42 fragments to Aβ40 fragments, A score of 0.35 or less indicates a predicted or diagnosed case of Alzheimer's disease. In some embodiments, the ratio of Aβ42 to Aβ40, or the Aβ40 fragment, is used. If the ratio of Aβ42 fragment to β is 0.3 or less, then Alzheimer's disease is Predicted or diagnosed as such. In some embodiments, Aβ40 versus Aβ4 The ratio of 2, or the ratio of Aβ42 fragment to Aβ40 fragment is 0.25 or so If the value is less than this, Alzheimer's disease is predicted or diagnosed. In terms of application methods, the ratio of Aβ42 to Aβ40, or the ratio of Aβ40 fragments to Aβ4 If the ratio of two fragments is 0.2 or less, Alzheimer's disease is predicted, It is diagnosed as such. In some embodiments, the ratio of Aβ42 to Aβ40, or A If the ratio of Aβ42 fragment to β40 fragment is 0.19 or less, Alzheimer's disease is predicted or diagnosed. In some embodiments, A The ratio of Aβ42 to Aβ40, or the ratio of an Aβ42 fragment to an Aβ40 fragment If the ratio is 0.18 or less, Alzheimer's disease is predicted or diagnosed as such. In some embodiments, if the ratio of Aβ42 to Aβ40, or the ratio of an Aβ42 fragment to an Aβ40 fragment is 0.17 or less, Alzheimer's disease is predicted or diagnosed. In some embodiments, if the ratio of Aβ42 to Aβ40 is 0.16 or less, Alzheimer's disease is predicted or diagnosed. In some embodiments, if the ratio of Aβ42 to Aβ40 is 0.15 or less, Alzheimer's disease is predicted or diagnosed. In some embodiments, if the ratio of Aβ42 to Aβ40 is 0.1 or less, Alzheimer's disease is predicted or diagnosed. In some embodiments, the method may include generating one or more precursor ions of Aβ or a fragment thereof. In some embodiments, at least one of the precursor ions has a mass / charge ratio of 1085.6 ± 0.5 or 1269.7 ± 0.5. In some embodiments, the method may include generating one or more fragment ions of Aβ or a fragment thereof. In some embodiments, at least one of the fragment ions is 812.37 ± 0.5, 869.4 ± 0.5, 968.43 ± 0.5, 86 If so, Alzheimer's disease is predicted or diagnosed.

[0086] In certain embodiments, the method includes generating one or more precursor ions of Aβ or a fragment thereof. In some embodiments, at least one of the precursor ions has a mass / charge ratio of 1085.6 ± 0.5 or 1269.7 ± 0.5. In some embodiments, the method may include generating one or more fragment ions of Aβ or a fragment thereof. In some embodiments, at least one of the fragment ions is 812.37 ± 0.5, 869.4 ± 0.5, 968.43 ± 0.5, 86 In some embodiments, at least one of the fragment ions is 812.37 ± 0.5, 869.4 ± 0.5, 968.43 ± 0.5, 86 In some embodiments, at least one of the fragment ions is 812.37 ± 0.5, 869.4 ± 0.5, 968.43 ± 0.5, 86 9.39±0.5, 968.44±0.5, 1067.5±0.5, or 1180.57 It has a mass / charge ratio of ±0.5.

[0087] In certain embodiments, the methods provided herein include adding internal standards. In some embodiments, the internal standard includes an isotope-labeled internal standard. In the embodiment, the internal standard is: 13 C 15 Includes N label. In some embodiments, internal label Jun is, 13 C 15 It contains at least one Phe, Leu, or Met labeled with N. In some embodiments, at least one of the internal standard precursor ions is 1110.7±0 It has a mass / charge ratio of 0.5. In some embodiments, the method is one of the internal standards or This may include generating multiple fragment ions. In some embodiments, the fragment At least one of the ment ions is 768.48±0.5, 825.5±0.5, or 8 It has a mass / charge ratio of 82.52 ± 0.5.

[0088] In certain embodiments, the limit of quantification of the method is less than or equal to 10 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 5 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 4 ng / mL. In that embodiment, the limit of quantification of the method is less than or equal to 3 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 2 ng / mL. In some embodiments, the limit of quantification of the method is less than or equal to 1 ng / mL. In the application method, the limit of quantification is less than or equal to 0.5 ng / mL. In the embodiments, the limit of quantification of the method is less than or equal to 0.2 ng / mL. In this embodiment, the limit of quantification of the method is less than or equal to 0.1 ng / mL.

[0089] In some embodiments, the detection limit of the method is less than or equal to 5 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 1 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.5 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.1 ng / mL. In some embodiments, the detection limit of the method is less than 0.05 ng / mL or equal to it. In some embodiments, the detection limit of the method is less than 0.01 ng / mL, or less than 0.01 ng / mL. It is equal to.

[0090] In some embodiments, amyloid beta is used as a derivative before mass spectrometry. It is not converted. In some embodiments, amyloid beta is converted by mass spectrometry. It is derivatized beforehand.

[0091] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is , cerebrospinal fluid (CSF). In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, The pull is either saliva or urine.

[0092] Suitable test samples include any test sample that may contain the target analyte. In some preferred embodiments, the sample is a biological sample; that is, any living It is a sample obtained from origin substances of organisms, such as animals, cell cultures, organ cultures, etc. Specific In a preferred embodiment, the sample is obtained from mammals such as dogs, cats, horses, etc. Particularly preferred mammals are primates, most preferably male or female humans. Particularly Preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or other tissue samples. Such samples can be obtained, for example, from patients; that is, living individuals who present themselves at the clinical site for the diagnosis, prediction, or treatment of a disease or condition, male or female. The test sample is preferably obtained from a patient, for example serum.

[0093] Sample preparation for mass spectrometry As methods that can be used to enrich amyloid beta more than other components (e.g., proteins) in the sample, for example, filtration methods, centrifugation methods, thin-layer chromatography (TLC) methods, electrophoresis methods including capillary electrophoresis, affinity separation methods including immunoaffinity separation methods, extraction methods including ethyl acetate extraction and methanol extraction, and the use of chaotropic agents, or any combination of the above, etc. can be mentioned.

[0094] The protein precipitation method is one preferred method for preparing the test sample. Such protein purification methods are well known in the art. For example, Polson et al., Journal of Chromatography B, Volume 785: pages 263 - 275 (20 03) describes protein precipitation techniques suitable for use in this method. The protein precipitation method leaves amyloid beta in the supernatant and removes most proteins from the sample. It can be used for removal. The sample separates the liquid supernatant from the precipitated protein. It can be centrifuged for this purpose. The resulting supernatant is then subjected to liquid chromatography and subsequent... It can be applied to mass spectrometry analysis. In certain embodiments, protein precipitation method, For example, by using methods such as acetonitrile protein precipitation, HPLC and mass spectrometry can be performed. Before performing treeing, highly turbulent liquid chromatography (HTLC) or other online Extraction becomes unnecessary. Therefore, in such embodiments, the method (1) aims (2) Perform protein precipitation of the sample; and (2) Online extraction or highly turbulent liquid Without using chromatography (HTLC), the supernatant can be converted to an HPLC-mass spectrometer. This relates to direct loading.

[0095] In some preferred embodiments, HPLC is used alone or for one or more purification processes. Used in combination with other methods to purify amyloid beta before mass spectrometry. It is possible. In such embodiments, the sample is placed in an HPLC extractor that captures the analyte. Extraction is performed using a tridge, then elution, and then chromatography is performed on a second HPLC column. It can be graphed or eluted onto an analytical HPLC column before ionization. The steps involved in the chromatography procedure can be linked together in an automated manner, so analysis The need for operator involvement during the material refining process can be minimized. Such characteristics result in time and cost savings, and also prevent operator errors. Opportunities can be eliminated.

[0096] For example, turbulence caused by HTLC columns and other methods can potentially enhance mass transfer rates. It has properties and is thought to improve separation characteristics. HTLC columns contain rigid particles. Components are separated by high-flow chromatography through a packed column. By using a high flow rate (e.g., 3-5 mL / min), turbulence is generated within the column, and the stationary phase and It causes nearly perfect interaction between the target analytes. In particular, high molecular weight species are not retained under turbulent conditions, and therefore interact with the biological fluid matrix. One advantage is that the accumulation of linked polymers is avoided. Multiple separation methods can be performed in a single procedure. In the combined HTLC method, the need for long sample preparation times is reduced, and the efficiency is significantly improved. It operates quickly. Such methods are superior to laminar flow (HPLC) chromatography. It also achieves separation performance. HTLC enables the direct injection of biological samples (plasma, urine, etc.). When injected directly, denatured proteins and other biological debris can rapidly disrupt the separation column. Because it blocks rapidly, this is difficult to achieve with conventional chromatography. Also, HTLC is less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, preferably 0 It also makes it possible to use very small sample volumes, such as 0.1 mL.

[0097] Examples of HTLC applied to sample preparation before analysis by mass spectrometry include: This is described separately. For example, Zimmer et al., J. Chromatogr. A854. See pages 23-35 (1999); U.S. Patent No. 5,968,367; and No. 5,91 See also No. 9,368; No. 5,795,469; and No. 5,772,874. Head In certain embodiments of the method, the sample is prepared as described above before being loaded onto the HTLC column. Protein precipitation treatment is performed; in an alternative preferred embodiment, the sample is subjected to protein precipitation. It can be directly loaded onto HTLC without any processing. The HTLC extraction column is preferred. The large particle column is a large particle column. In various embodiments, one or more steps of the method are This can be carried out in an online, automated manner. For example, in one embodiment, steps (i) to (v) are performed using an online automated method. In other cases, ionization and The detection steps are performed online after steps (i) to (v).

[0098] Liquid chromatography (LC), including high-performance liquid chromatography (HPLC), It is based on relatively low-speed laminar flow technology. Conventional HPLC analytical methods use a sample passing through a column. The laminar flow of the column is the basis for separating the target analyte from the sample, and depends on the column packing material. Those skilled in the art understand that separation in such a column is a diffusion process. HPLC is applicable without problems to the separation of compounds in biological samples, but a considerable amount of saturates... Sample preparation is required before separation and subsequent analysis by mass spectrometry (MS). This technology is labor-intensive. Furthermore, most HPLC systems use mass spectrometry. The equipment is not being used to its full potential, and one HPLC system is being used as one MS instrument. It merely allows connection, and as a result, it takes redundant time to perform numerous assays. It is necessary.

[0099] Regarding the use of HPLC for sample removal before mass spectrometry analysis, Various methods are described. For example, Taylor et al., Therapeutic Dr. ug Monitoring, Vol. 22: pp. 608-6012 (2000); and Salm Ra, Clin.Therapeutics, Vol. 22 Supl.B: pp. B71-B85 (2 See (Year 000).

[0100] Those skilled in the art will select an HPLC instrument and column suitable for use with amyloid beta. It is possible. Chromatographic columns are media that facilitate the separation (i.e., fractionation) of chemical parts. It generally contains a body (i.e., a filler). Fine particles can be cited as a medium. The particles possess a bonding surface that interacts with various chemical parts, promoting the separation of those chemical parts. One suitable bonding surface is a hydrophobic bonding surface, such as an alkyl bonding surface. The bonding surface is a C-4, C-8, C-12, or C-18 bonded alkyl group, preferably C- It may contain 18 binding groups. The chromatographic column has an injection port for receiving the sample. It has a drain port for discharging the effluent containing the fractionated sample. In this application method, the sample (or pre-purified sample) is injected into the column at the injection port. It is applied, eluted with a solvent or solvent mixture, and discharged through the discharge port. The solvent mode can be selected to elute the target analyte. For example, liquid chromatography Tography uses gradient modes, isocratic modes, or polymorphic (i.e., mixed) modes. This can be carried out using a chromatograph. During the chromatography period, the separation of materials is subject to varying factors. For example, the selection of the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, and the temperature. It is affected by, among other things.

[0101] In certain embodiments, the analyte is reversibly preserved by the column packing material. Under conditions where one or more other substances are retained while the sample is placed in a column, It can be purified by applying it to. In such embodiments, the conditions of the first mobile phase are The target analyte can be configured to be retained by the column, and the second mobile phase The condition is that after the substances that were not retained are washed out, the retained substances are removed from the column. It can be configured to be extracted. Alternatively, the analyte may be one or more other substances. In comparison, under mobile phase conditions where the target analyte elutes at a different rate, the sample It can be purified by applying it to a column. Such a procedure can be performed on one or more samples. Potential to enrich the quantity of one or more target analytes compared to other components of the number. There is.

[0102] In one preferred embodiment, HTLC is a hydrophobic column chromatography system. This can be followed by the HPLC described above. In certain preferred embodiments, Cohesive Tech TurboFlow Cyclone P® polymer manufactured by nologies Inc. A base column (particle size 60 μm, column dimensions 50 × 1.0 mm, pore size 100 Å) is used. Used. In a related preferred embodiment, Phe has hydrophilic end capping. Synergi Polar-RP® ether manufactured by nomenex Inc. Conjugated phenyl analysis column (particle size 4 μm, column dimensions 150 × 2.0 mm, pore size) 80 Å) is used. In certain preferred embodiments, HTLC and HPLC are used as the mobile phase. This is carried out using HPLC-grade ultrapure water and 100% methanol.

[0103] Careful valve selection and connector piping work eliminate the need for any manual steps. , two or more chromatography images are used to allow a substance to pass from one column to the next. - Columns may be connected as needed. In preferred embodiments, valve selection and piping The work is managed by a computer pre-programmed to perform the necessary steps. It is considered. Most preferably, the chromatography system also includes a detector system, for example, M The S system is connected in such an online manner. Therefore, the operator is Sun All you need to do is place the pull tray into the autosampler, and the rest of the work will be done by computer. - The process is carried out under controlled conditions, and as a result, the purification and analysis of all selected samples are completed. .

[0104] In certain preferred embodiments, amyloid beta or its fragments in the sample are It can be purified before ionization. In a particularly preferred embodiment, chromatography is performed. This is not schromatography.

[0105] Detection and quantification by mass spectrometry In various embodiments, amyloid beta or its fragments are known to those skilled in the art. It can be ionized by any method. Mass spectrometry is used to ionize the fractions. It features an ion source that ionizes the sample and generates charged molecules for further analysis. This is performed using a mass spectrometer. For example, sample ionization is performed using electron ionization, etc. Chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization Photoionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atomic impact (FAB) Liquid secondary ionization (LSI), Matrix-assisted laser desorption / ionization (MALDI) Field ionization, field desorption, thermospray / plasma spray ionization, surface enhancement laser Zer desorption ionization (SELDI), inductively coupled plasma (ICP), and particle beam ion This can be carried out by ionization. Those skilled in the art will know that the choice of ionization method depends on the analyte being measured, the sample. This can be determined based on factors such as the type of detector, the selection of positive and negative modes, etc. To understand it as such.

[0106] In a preferred embodiment, amyloid beta or its fragments are positive mode Alternatively, ions can be ionized by heated electrospray ionization (HESI) in negative mode. In an alternative embodiment, amyloid beta or its fragments are positively modified. Electrospray ionization (ESI) in negative or neutral mode or atmospheric pressure chemical ionization It is ionized by ON activation (APCI).

[0107] After the sample is ionized, the resulting positively charged or negatively charged ions Ions can be analyzed to determine the mass-to-charge ratio. Suitable analyzers include quadrupole analyzers, ion trap analyzers, And time-of-flight analyzers are also mentioned. Ions are detected using several detection modes. It is possible. For example, selected ions, i.e., selective ion monitoring mode Is it detectable using a SIM, or is the ion in scanning mode? For example, using multiple reaction monitoring (MRM) or selective reaction monitoring (SRM). It can be detected using a quadrupole analyzer. Preferably, the mass-to-charge ratio is detected using a quadrupole analyzer. This is determined by the following: For example, in a "quadrupole" or "quadrupole-type ion trap" device, between the electrodes The applied DC potential, the amplitude of the RF signal, and a force proportional to the mass / charge ratio are oscillating. Applied to ions in a radio frequency field. The voltage and amplitude are applied to ions with a specific mass / charge ratio. Only the ON ions are selected to move along the quadrupole length, while all other ions are selected to disengage. It is possible. Therefore, a quadrupole-type device is a "mass filter" for ions injected into the device. It can function as both a "-" and a "mass detector".

[0108] By utilizing "tandem mass spectrometry" or "MS / MS," the resolution of MS technology can be improved. This technology can enhance the effect. In this technology, a precursor ion (parent ion) generated from the target molecule is used. The precursor ions (also called ON) can be filtered within the MS instrument, and It is then fragmented and one of the fragments is then analyzed in the second MS procedure. Alternatively, it can become multiple fragment ions (also called daughter ions or product ions). By carefully selecting precursor ions, only the ions produced from a specific analyte can be identified. It passes through a composting chamber, where it collides with atoms of an inert gas and forms fragment ions. It produces ions. Both the precursor ion and the fragment ion undergo ionization / fragmentation. Because it produces reproducibly under a series of predetermined conditions, MS / MS technology is an extremely powerful technology. It can provide analytical tools. For example, a combination of filtering / fragmentation can detect interfering substances. It can be used to remove [unclear], and is particularly useful in composite samples, such as biological samples. It may be useful.

[0109] Mass spectrometers generally perform ion scanning; that is, scanning within a predetermined range (e.g., 100°). In a range of ~1000 amu, the relative abundance of each ion with a specific mass / charge is given by Eugen. Provided to the user. The results of the analyte assay, i.e., the mass spectrum, are in the art. By a great many known methods, it is possible to correlate the amount of the analyte in the original sample with the amount of the analyte. This is possible. For example, assuming that sampling and analysis parameters are carefully controlled. Then, the relative abundance of a given ion is converted to the absolute amount of the original molecule. It can be compared with the table. Alternatively, molecular standards can be run along with the sample. This is done, and then a standard curve is constructed based on the ions generated from that standard. Using a standard curve, the relative abundance of a given ion is converted to the absolute amount of the original molecule. It is possible. In certain preferred embodiments, an internal standard calculates the amount of amyloid beta. Used to generate a standard curve for. Method for generating and using such a standard curve. This is well known in the art, and those skilled in the art have the ability to select appropriate internal standards. For example, amyloid-beta isotopes can be used as internal standards. There are many other methods for relating to the amount of original molecules, which are well known to those skilled in the art. .

[0110] One or more steps of the method may be carried out using automated machinery. In the embodiment, one or more purification steps are performed online, and more preferably Furthermore, all purification and mass spectrometry steps are performed online. obtain.

[0111] In certain embodiments, for example, precursor ions are isolated for the purpose of further fragmentation. In MS / MS, collision activation dissociation is used. It is often used to generate fragment ions for further detection. In D, the precursor ions gain energy through collisions with the inert gas, but then " Fragmentation occurs through a process called "single-molecule decomposition reaction." Increase in vibrational energy. As a result, sufficient energy is generated so that certain bonds within the ion can be broken, and the precursor It must be stored within the ON timeframe.

[0112] In a particularly preferred embodiment, amyloid beta is obtained using MS / MS as follows: The sample is detected and / or quantified by liquid chromatography, preferably HP. Processed by LC, the liquid solvent flow from the chromatographic column is analyzed by MS / MS analysis. The solvent / analyte mixture enters the heated nebulizer interface of the ser and then The analyte is converted into vapor within the heated tube of the interface. It is ionized by an ionization device. The ions, such as precursor ions, pass through the opening of the device. Then, it enters the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters. - and based on its mass-to-charge ratio (m / z), ions (i.e., "precursors" and " This allows for the selection of "fragment" ions. Quadrupole 2 (Q2) is a collision cell, and there The ions are then fragmented. The first quadrupole (Q1) of the mass spectrometer is used for amyloid Select the molecule using the mass-to-charge ratio of beta. Correct mass / charge ratio for amyloid beta. Precursor ions having a ratio can be introduced into the collision chamber (Q2), while other qualities Any undesirable ions with a quantity / charge ratio will collide with the sides of the quadrupole and be removed. The precursor ions entering Q2 collide with neutral argon gas molecules and become fragmented. This process is called collision-activated dissociation (CAD). The resulting fragment ions It is introduced into quadrupole 3 (Q3), where amyloid-beta fragment ions are produced. While some ions are selected, others are removed.

[0113] This method is performed using MS / MS in either positive or negative ion mode. This may be related. Using a standard method well known in the art, a person skilled in the art can construct a quadrupole 3 (Q3 ) one or more of the specific amyloid beta precursor ions available in the selection in ) It has the ability to distinguish between different ions.

[0114] When amyloid beta precursor ions contain an alcohol or amine group, generally, the precursor Fragment ions corresponding to ion dehydration or deamination are formed. In the case of a precursor ion containing a hol group, such a fragment ion formed by dehydration is , caused by the loss of one or more water molecules from the precursor ion (i.e., precursor ion The difference in mass-to-charge ratio between ON and fragment ions is approximately 18 for the loss of one water molecule. (For example, the loss of two water molecules is approximately 36.) Such fragment ions formed by synthesis and deamination are one or more ammonia. This is caused by the loss of near molecules (i.e., between the precursor ion and the fragment ion). The difference in the mass-to-charge ratio is approximately 17 for the loss of one ammonia molecule, or ammonia The loss of two molecules is approximately 34, etc. Similarly, one or more alcohols and amino acids Precursor ions containing the ¹ group typically contain one or more water molecules and / or one or more The loss of ammonia molecules forms fragment ions (i.e., precursor ions). The difference in the mass-to-charge ratio between the fragment ion and the water molecule is due to the loss of one water molecule and the ammonia. (Approximately 35 for the loss of one molecule). Generally, the dehydration or deamination of the precursor ion is The fragment ions in question are not specific fragment ions for a particular analyte. Therefore, in a preferred embodiment of the present invention, MS / MS is used to remove at least amyloid beta Another fragment ion was detected, which is one or more water molecules separated from the precursor ion. The only molecule lost, and / or the only one or more ammonia molecules lost. It will be implemented in a way that does not fall under category 1.

[0115] When an ion collides with a detector, the ion is converted into a pulse of electrons, which is then converted into a digital signal. It produces [something]. The acquired data plots the count of collected ions against time. The data is transmitted to a computer. The resulting mass chromatogram is obtained using a conventional HPLC method. It is similar to the chromatogram produced by [the following method]. The area under the peak corresponding to a specific ion, The amplitude of such peaks is measured, and the area or amplitude is determined to be the amount of the target analyte. Associated. In certain embodiments, relating to fragment ions and / or precursor ions. The area under the curve or the amplitude of the peak is measured to determine the amount of amyloid beta. As described above, calibration standards are based on peaks derived from one or more ions of the internal molecular standard. Using a curve, the relative abundance of a given ion is converted to the absolute amount of the original analyte. It is possible.

[0116] The following examples serve to illustrate the present invention. These examples cover the scope of this method. There is absolutely no intention to impose any restrictions. [Examples]

[0117] [Example 1] Pre-treatment of the device and stabilization of amyloid beta Pre-treatment of 96-well polypropylene plates and microcentrifuge test tubes E. coli was lysed in a 96-well plate and then precipitated using methanol.

[0118] Next, the methanol is discarded, while the pellet is resuspended in ammonium bicarbonate at pH 8. did.

[0119] Bovine trypsin from Sigma was added, and the sample was incubated at 60°C for 2 days. I did it.

[0120] Next, all digested material is discarded for disposal purposes, or used to pre-treat pipette tips. I pooled my money.

[0121] The empty plates and test tubes were completely dried.

[0122] Pre-treatment of pipette tips Using a pipette tip, pipette and dispense the E. coli digest at least three times. Next, the samples were incubated at 60°C for two days, or until completely dry.

[0123] Stabilization of amyloid-beta peptide AB40, AB42, and the internal standard were synthesized in a pre-treated test tube, and then 4 mg of The suspension was resuspended in 6M urea containing BSA.

[0124] The standard then includes N-terminal and C-terminal antibodies, as well as apolipoproteins E2 and E4. Each was diluted 1:10 in 0.1M PBS.

[0125] Each mixture was incubated in a shaker at room temperature for 1 hour.

[0126] Next, mix AB40 and AB42, and before freezing, add 0.1M PBS and 0 A high-value calibrator standard was prepared by diluting in 0.4 mg / mL BSA.

[0127] The table below shows a left-right comparison of the same sample prepared in treated and untreated vials. show.

[0128] [Table A]

[0129] [Example 2] Sample preparation Using a pre-treated pipette tip, pre-treat 0.5 mL of the sample or standard. The sample was pipetted into a 96-well plate.

[0130] 5 ng of internal standard was added.

[0131] 250 μL of 18M urea was added.

[0132] 2ug of Lys-C was added to each sample to digest amyloid beta.

[0133] Before digesting in a microwave with enzymes at 450W, 45℃ for 4 hours, seal with an adhesive lid. The box was sealed.

[0134] Next, the samples were extracted using a water-mixed strong anion exchange mode.

[0135] Next, the sample eluate was completely dried using heated nitrogen.

[0136] Next, the samples were resuspended in a reconstitution buffer for LC-MS / MS analysis.

[0137] [Example 3] Detection and quantification of amyloid-beta fragments by MS / MS. Ions are introduced into the first quadrupole (Q1), and Aβ40 fragments and Aβ42 fragments are introduced. For each of the points, 1085.6±0.5m / z or 1269.7±0.5m / z Ions were selected using one of the following mass-to-charge ratios. The ON ions collided with argon gas, generating ionic fragments, but for further selection... Then, an ion fragment was introduced into quadrupole 3 (Q3). Simultaneously, an isotope-diluted mass spectrum was used. The same process using trometry was performed using internal standards. The following mass transitions were observed. This was used for detection and quantification during the validation period under positive polarity.

[0138] [Table 1]

[0139] Examples of patient chromatograms are shown in Figures 6 and 7.

[0140] By comparing various extraction methods, we optimized the recovery rate of amyloid-beta fragments.

[0141] [Table 2] Waters MAX: Mixed strong anion exchange mode → For validating the final assay. use Waters MXC: Mixed strong cation exchange mode Waters HLB: Silica fabricated for hydrophobic compounds Agilent C18: A typical C18 filler PhenomenexCX: Mixed strong cation exchange mode ThermoSAX: Strong anion exchange

[0142] The mixed strong anion exchange mode offers the best recovery rate for amyloid-beta fragments. This brought about...

[0143] The urea present in the digest increases the recovery rate of the analyte. The following values ​​were determined:

[0144] [Table 3]

[0145] We compared the sensitivity of C4 and C8 analytical columns. The C4 column showed superior sensitivity. However, in this technical field, C8 and C18 columns are generally preferred. That was unexpected. Figure 11.

[0146] [Example 4] Reportable range and linearity of the assay To demonstrate the linearity of amyloid beta detection in the assay, a zero standard was used. A single blank and a spiked standard were prepared and analyzed. Five consecutive runs were performed. From the quadratic regression derived from, a correlation coefficient of 0.995 or greater with ±20% accuracy is obtained. The obtained and quantifiable linear range was found to be in the range of 0.1 to 25 ng / mL. (Figure) Figures 4 and 5.

[0147] [Example 5] Precision of the assay The intra-assay reproducibility of the samples was tested against the following quality control levels:

[0148] [Table 4]

[0149] Precision during the run: 10 repeated measurements of each quality control within a single assay. The analysis was conducted in the following order: low, medium, and high.

[0150] Pass / fail criteria: %CV should be less than the acceptable value ≤TEa / 2. The TEa of I has been determined to be 30%.

[0151] The %CV of beta-amyloid-40 was 14 at all three quality control levels. The range was 0.31% to 6.55%.

[0152] The %CV of beta-amyloid-42 was 14 at all three quality control levels. The range was 0.93% to 7.51%.

[0153] Overall precision: Inter-assay validity data was analyzed.

[0154] Pass / fail criteria: If total SD ≥ 1 / 2TEa, it is not acceptable, or the total SD is the defined maximum. It must be less than the large SD or CV.

[0155] %CV should be less than the acceptable value ≤TEa / 2. TEA for this assay It has been determined to be 30%.

[0156] The %CV of beta-amyloid-40 was 10 at all three quality control levels. The range was 0.21% to 4.28%.

[0157] The %CV of beta-amyloid-42 was 14 at all three quality control levels. The range was 0.63% to 8.17%.

[0158] [Example 6] Analytical sensitivity (detection limit) Blank Limit (LOB): Calculation: LOB = Average Blank Value + 2SD.

[0159] 21 matrix blanks were analyzed within a single assay, and then the inverse values ​​were calculated. This is used to calculate the following LOBs.

[0160] LOB of AB40: 31.32 pg / mL.

[0161] LOB of AB42: 32.11 pg / mL.

[0162] [Table 5]

[0163] Limit of Detection (LOD): Calculation: LOD = Mean value of blanks + 4SD.

[0164] Twenty-one matrix blanks were analyzed within a single assay, and then, the inverse values ​​were calculated. This is used to calculate the following LOD.

[0165] LOD of AB40: 77.78 pg / mL.

[0166] LOD of AB42: 74.21 pg / mL.

[0167] [Table 6]

[0168] Limit of Quantitative The lowest concentration equivalent to that.

[0169] The limit of quantification for both beta-amyloid 40 and 42 has been determined to be 100 pg / mL. ru.

[0170] [Table 7]

[0171] [Table 8]

[0172] [Table 9]

[0173] [Table 10]

[0174] [Example 7] Analyte measurement range (AMR) Pass / fail criteria: When TEa is 30%, the deviation of the average of the measured values ​​from the predicted range is... The coefficient of variation should be less than or equal to 2SD or 20%CV.

[0175] The %CV of beta-amyloid-40 is in the range of 6.52-12.01%, which is acceptable. It is considered to be.

[0176] The %CV of beta-amyloid-42 is in the range of 6.27–14.24%, which is acceptable. It is considered to be.

[0177] [Table 11]

[0178] [Example 8] Accuracy Recovery rate of known standards Pass / fail criteria: Errors resulting from inability to completely recover (recovered amount - added amount) are considered to be TEa. At 30%, the CV should be less than or equal to 2SD or 15%.

[0179] Six strip bovine cerebrospinal fluid samples were prepared at the following concentrations: 1000, 2000, and 3000. The test was spiked at 4000, 8000, and 9000 pg / mL, with each spike level being tripled. The assay was performed.

[0180] Next, six different spike levels were used, with stripped bovine cerebrospinal fluid as a diluent. The solution was then diluted in ratios of 1:3 and 1:5, and the assay was performed again in triplicate.

[0181] [Table 12]

[0182] [Table 13]

[0183] [Table 14]

[0184] [Table 15]

[0185] [Table 16]

[0186] [Table 17]

[0187] [Example 9] Sample stability Pass / fail criteria: When TEa is equal to 30%, between the baseline value and the time / temperature sample value. The sample is considered stable as long as the difference in mean values ​​is ≤TEa / 2 with respect to the analyte. .

[0188] Freeze / thaw stability: Freeze-thaw analysis was performed on a pool of 6 patients divided into 4 aliquots. This was done by analyzing all four aliquots in each patient pool, from -60 to - It was frozen at 80°C. The second to fourth aliquots were thawed at ambient temperature of 18-26°C and then frozen. The third and fourth aliquots were thawed at an ambient temperature of 18-26°C and then frozen. The fourth aliquot was then thawed at an ambient temperature of 18-26°C and then frozen.

[0189] Finally, all aliquots were thawed at ambient temperature of 18-26°C, and then technically triple-layered. The analysis was performed using the following method. The freeze-thaw analysis includes data related to three types of freeze-thaw cycles.

[0190] Beta-amyloid 40 and beta-amyloid 42 can withstand up to two freeze-thaw cycles. It satisfies acceptable stability.

[0191] [Table 18-1] [Table 18-2] [Table 18-3]

[0192] Stability of the sample after extraction: As a baseline value, on the same day as the sample extraction, 1 Zero samples were analyzed. The following day, the same samples were reinjected and compared with baseline values. Ta.

[0193] Beta-amyloid 40 and 42 are present in the C-stack of a CTC autosampler. The sample shows post-extraction stability for up to 1 day at 4°C.

[0194] [Table 19]

[0195] Refrigeration stability (2.0~8.0℃): The sample remains stable even when refrigerated for up to 5 days. This demonstrated that.

[0196] [Table 20]

[0197] [Table 21]

[0198] Room temperature stability (18.0~26.0℃): The sample is stable for up to 3 days at 18~26℃. be.

[0199] [Table 22]

[0200] [Table 23]

[0201] Freezing stability (-10.0 to -30.0°C): The sample can be frozen for up to 31 days at -10 to -30°C. It remains stable for several days.

[0202] [Table 24]

[0203] [Table 25]

[0204] [Example 10] Interference Test Pass / fail criteria: Differences due to potential interfering substances should be ≤2SD, or 20%. CV is considered acceptable.

[0205] Interference due to hemolysis: Six spike pools, baseline, small, medium, and large We analyzed the interference caused by simulated hemolysis in a three-part series.

[0206] Beta-amyloid-40 and beta-amyloid-42 were found in non-hemolytic and small-scale hemolytic cerebral vertebrae. Only cerebrospinal fluid samples are accepted.

[0207] [Table 26]

[0208] [Table 27]

[0209] [Table 28]

[0210] [Table 29]

[0211] Interference due to lipidemia: Six spike pools, baseline, small, medium, and We conducted a triplicate analysis of the interference with large-scale lipidemia.

[0212] Beta-amyloid-40 and beta-amyloid-42 are associated with small and moderate cases of cerebrospinal fluid retention. Cerebrospinal fluid samples are acceptable.

[0213] [Table 30]

[0214] [Table 31]

[0215] [Table 32]

[0216] [Table 33]

[0217] Bilirubin interference: Six spike pools, baseline, small, medium, and We analyzed the disruption caused by widespread jaundice in three separate studies.

[0218] Beta-amyloid-40 and beta-amyloid-42 are associated with the brain and spinal cord in patients with no jaundice and mild jaundice. Cerebrospinal fluid samples are acceptable.

[0219] [Table 34]

[0220] [Table 35]

[0221] [Table 36]

[0222] [Table 37]

[0223] [Example 11] Ion suppression Ten patient samples were selected.

[0224] To monitor ion suppression across the entire gradient, open the acquisition window for up to 10 minutes. Released. Ten samples were injected through the analysis column, while beta-amyloid 40 and A mixture of 42 digested peptides was injected into the post-column.

[0225] When the internal standard AB40 or AB42 is eluted, the total ions of AB40 or AB42 are detected. If the chromatogram (TIC) shows a decrease of ≥15% in signal intensity, Ion repression is determined to be present in the assay.

[0226] The TIC of digested peptides AB40 and AB42 is suppressed within the gradient during analyte elution. It did not show this. The TIC signal intensity was constant, and the difference in signal intensity was ≤15%. This indicates that the assay parameters are within the acceptable range.

[0227] [Example 12] Carryover After analyzing the high-value calibrator standard, we then analyzed the four matrix blanks. This sequence was repeated two more times. The matrix blank after high-value calibrator analysis was averaged. Based on the average calculated concentration, the recovery rate for both beta-amyloid 40 and 42 was 0.06%. be.

[0228] No carryover was observed in this assay.

[0229] [Table 38]

[0230] [Example 13] Reference interval (RI) Beta-amyloid 40: 6000.00~15000.00 pg / mL

[0231] Beta-amyloid 42: 700.00~4000.00 pg / mL

[0232] [Example 14] Alzheimer's disease patient data Cerebrospinal fluid (C) of 211 subjects, including patients diagnosed with Alzheimer's disease and healthy controls. We analyzed the SF (Science Fiction). See Figures 8-10.

[0233] Aβ40 and Aβ42 were detected in all CSF samples. Surprisingly. Aβ42 levels were approximately 10 times higher than the publicly reported levels. The pull rate was in the range of 1-8 ng / mL. Alzheimer's disease patients have Aβ42:Aβ4 Based on the fact that the ratio of 0 is lower compared to borderline patients and healthy subjects (highest ratio) They were distinguishable. Figure 8.

[0234] [Example 15] Additional recovery tests Sample preparation: All plastic disposable tools are pre-treated and non- This technology prevents specific binding and stabilizes the Aβ standard, thereby enhancing long-term storage stability.

[0235] A strong protein denaturant was added to human CSF (500uL), and the sample was then tanned. The protein was digested and then subjected to solid-phase extraction. The sample was processed on a robotic liquid handler (Hamilton Microlab Star A857) connected to a CEREX IP8 (SPEware) solid-phase extraction manifold.

[0236] Separation: HPLC separation was performed on an Aria TLX-4 system (Thermo Scientific) using a Waters XBridge Protein BEH C4 column, 4.6 × 100 mm, 3.5 micron, 300 Å.

[0237] Detection: A Thermo TSQ Quantiva triple quadrupole mass spectrometer

[0238] The linearity data showed an R value of at least 0.98 for both peptides and a %CV ≤ 15% throughout the eight calibration standards. 2

[0239] The limit of quantification was 100 pg / mL for both peptides.

[0240] For Aβ40 and Aβ42, the assay precision (%CV) was ≤ 15% and the recovery rate was in the range of 84% - 112%.

[0241] Precision and accuracy within the assay (N = 10) and between assays (N = 5, over 5 days)

[0242]

Table 39

[0243] Stability of the assay over 8 months [[ID=​​​​​​

[0245] This technology enables the stabilization of Aβ peptides and facilitates the long-term storage of calibrators and quality control standards. This made it possible.

[0246] The frozen calibrator remained stable for at least 8 months when stored at -80°C. .

[0247] The stabilization measures eliminated nonspecific bonding and resulted in high analyte recovery rates. Figures 12 and 13.

[0248] Patient CSF Aβ42 concentration values ​​are better measured using LC-MS / MS assays than ELIS. This was higher than when using assay A. This is a comparison of the two methodologies. This is consistent with the report. Future research may explain the differences by showing that the pre-analysis factors described herein explain the differences. It is expected that this will clarify whether it will be useful or not.

[0249] [Table 41]

[0250] Conclusion: This novel approach addresses one of the key challenges in the quantification of Aβ40 and Aβ42. : Reproducibility issues caused by non-specific coupling were eliminated.

[0251] [Example 16] Patient diagnostic testing Three quality controls (low, medium, high) are run, and three different calibration curves are followed. The points were displayed. QC ensured accurate quantification throughout the plate at the start of the run. Running was performed at the start and end times. The accuracy of the quality control is as follows:

[0252] [Table 42]

[0253] Patient sample ranges of Aβ40 and 42 Based on 72 patient samples. Aβ40: 5135.33 - 25348.94 pg / mL Aβ42: 1068.00 - 5499.76 pg / mL.

[0254] Normalize different levels of Aβ40 and 42.

[0255] Due to plaque formation or insufficient washing, the value of Aβ42 decreases, so the ratio of Aβ42 / 4 0 also decreases.

[0256] The levels of Aβ40 and 42 seem to increase / decrease independently of each other. <000199-eight><000199-nine>Sort the data by the ratio of Aβ42 / 40 and divide it into two sets based on the median (Aβ42 / 40 = 0.17).

[0258] Half of the data was divided as the median of <Aβ42 / 40.

[0259] Half of the data was divided as the median of >Aβ42 / 40.

[0260] Calculated the mean, standard deviation, and %CV.

[0261] When the ratio value was the median of <Aβ42 / 4, numerical values ≥ 1SD were removed.

[0262] When the ratio value was the median of >Aβ42 / 40, numerical values ≤ 1SD were removed.

[0263] Values outside 1SD were classified as "borderline". <​This specification includes all documents, patents, and patent applications, as well as all other documents, that are described or cited herein. And the content of the electronically available information is incorporated as references in the individual public documents. To the same extent as it is specifically and individually suggested, it is incorporated herein by reference in its entirety. Applicants may use any such documents, patents, patent applications, or other physical and electronic documents. We reserve the right to physically incorporate any and all materials and information derived from this document into this application. do.

[0265] The methods described herein by example may not apply to any single element not specifically disclosed herein. Alternatively, it can be suitably implemented even without multiple elements, one or more limitations. For example, terms such as "comprising," "including," and "containing" The terms "ining," etc., must be interpreted broadly and non-limitingly. Furthermore, the terms adopted in this specification The terms and expressions used are intended to be descriptive, not restrictive. Furthermore, in the use of such terms and expressions, any equivalent of the presented and described characteristics There is no intention to exclude or exclude any part thereof. Various modifications are within the scope of the claimed invention. It is recognized that this is possible. Therefore, the present invention relates to preferred embodiments and optional characteristics. Modifications of the Invention that are incorporated into the Invention, as specifically disclosed herein, are also disclosed herein. And changes are understood to be implementable by those skilled in the art, and such modifications and changes are It should be understood that this falls within the scope of the present invention.

[0266] The present invention is described extensively and comprehensively herein. Within the scope of the general disclosure Each of the narrower species and subgenus groupings included also forms part of this method. This is a general description of a method that involves a condition or negative limitation for removing any subject from a genus. The extracted material may include, whether or not it is specifically listed herein. ru.

[0267] Other embodiments are included in the following claims. Furthermore, features or aspects of the method. However, if described by the Markush group, a person skilled in the art will know that the invention is thus, This describes either individual members or subgroups of members of the Kush group. I recognize that it exists.

Claims

1. A method for determining the amounts of amyloid beta 40 (Aβ40) and amyloid beta 42 (Aβ42) in a sample, (a) Digesting the amyloid beta in the sample to produce Aβ40 containing the sequence GAIIGLMVGGVV (SEQ ID NO: 2) and Aβ42 containing the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4), (b) Purify Aβ40 and Aβ42. (c) Ionizing Aβ40 and Aβ42 to produce precursor ions, and (d) generating one or more fragment ions of Aβ40 and Aβ42, and (e) Determine the amount of one or more fragment ions from step (c) or (d) or both by mass spectrometry. A method comprising, wherein one or more fragment ions include ions having a mass / charge ratio selected from 812.37±0.5, 869.4±0.5, 968.43±0.5, 869.39±0.5, 968.44±0.5, 1067.5±0.5, or 1180.57±0.

5.

2. The method according to claim 1, wherein the purification includes liquid chromatography.

3. The method according to claim 2, wherein the liquid chromatography includes high-performance liquid chromatography (HPLC).

4. The method according to claim 1, further comprising a C-4 analytical column.

5. The method according to claim 1, further comprising incubating the sample with an agent that stabilizes amyloid beta.

6. The method according to claim 5, wherein the agent comprises an antibody that binds to the C-terminus of amyloid beta, an antibody that binds to the N-terminus of amyloid beta, apolipoprotein E2, apolipoprotein E4, or a combination thereof.

7. The method according to claim 5, wherein the agent provides stability over at least three freeze-thaw cycles.

8. The method according to claim 1, wherein the agent provides stability at -70°C for at least two months.

9. The method according to claim 1, comprising mixed-mode anion exchange extraction.

10. The method according to claim 1, wherein the ionization includes heated electrospray ionization.

11. The method according to claim 1, wherein the ionization includes ionization in positive mode.

12. The method according to claim 1, wherein the generation of the fragment ions includes using collision energies of 20V to 45V.

13. The method according to claim 1, wherein the precursor ion has a mass / charge ratio of 1085.6 ± 0.

5.

14. The method according to claim 1, further comprising adding an internal standard.

15. The method according to claim 14, wherein the internal standard is labeled with an isotope.

16. The aforementioned internal standard, １３ C １５ The method according to claim 15, including an N label.

17. The method according to claim 15, wherein the precursor ion of the internal standard has a mass / charge ratio of 1110.7 ± 0.

5.

18. The method according to claim 15, wherein the one or more fragment ions of the internal standard have a mass / charge ratio of 768.48 ± 0.5, 825.5 ± 0.5, or 882.52 ± 0.

5.

19. The method according to claim 1, wherein the limit of quantification of the method is less than or equal to 10 ng / mL.

20. The method according to claim 1, wherein the sample is cerebrospinal fluid.

21. The method according to claim 1, wherein the digestion is carried out by LysC.

22. The method according to claim 21, further comprising adding urea to the digestion.

23. The method according to claim 21, wherein the digestion further comprises digestion in microwaves.

24. The method according to claim 1, wherein the Aβ40 further comprises a flying peptide.

25. The method according to claim 24, wherein the flying peptide is hydrophilic.

26. The method according to claim 24, wherein the flying peptide comprises one or more N-terminal or C-terminal amino acid residues.

27. ​​The method according to claim 1, further comprising pre-treating the surface of an instrument that comes into contact with a sample with an agent that prevents amyloid beta from adhering to the surface.

28. The method according to claim 27, wherein the drug is a lysate of E. coli.

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