Detection of apolipoprotein e isotypes by mass spectrometry

Abstract

To provide a method for determining an apolipoprotein E (APOE) phenotype in a sample by mass spectrometry.SOLUTION: There is provided a method including: (a) purifying ApoE in a sample; (b) ionizing the ApoE in the sample to generate one or more ions of ApoE; and (c) detecting ions derived from step (b) by mass spectrometry, in which an ApoE allele present in the sample is determined from the identity of the ions detected in step (c). In another aspect, there is provided a method for diagnosing or predicting Alzheimer disease or dementia.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates to the detection or quantification of apolipoprotein E. In certain embodiments, the present invention The present invention is directed to the detection of apolipoprotein E or its alleles by mass spectrometry. This relates to a method for [Background technology]

[0002] Alzheimer's disease is the most common form of dementia affecting the elderly population. Lelzheimer's disease is characterized by a progressive decline in cognitive abilities, particularly memory and learning. Apolipoprotein E (APOE) is associated with a significant increase in the expression of APOE at the onset of Alzheimer's disease The human APOE gene has three polymorphic alleles, ε2, ε3, and ε4, resulting in The result is six different phenotypes: ε2 / ε2, ε2 / ε3, ε3 / ε3, ε2 / ε4, and ε3 / ε4. , and ε4 / ε4.

[0003] The accuracy and sensitivity of current clinical diagnostic methods for predicting or diagnosing Alzheimer's disease are Low. Accurate and sensitive assays for detecting apolipoprotein E are needed. In particular, accurate and sensitive assays for detecting various isoforms are needed. do. Summary of the Invention [Problem to be solved by the invention]

[0004] As used herein, mass spectrometry, such as tandem mass spectrometry, is used to analyze the sample. A method for detecting or determining the amount of apolipoprotein E (APOE) in a sample is provided. [Means for solving the problem]

[0005] In certain embodiments, the method described herein for detecting or determining the amount of apolipoprotein E is The provided method includes: (a) purifying apolipoprotein E in a sample; and (b) (c) ionizing apolipoprotein E in said sample; and and detecting or determining the amount of apolipoprotein E ions by immunoassay. In this case, the amount of apolipoprotein E ions is proportional to the amount of apolipoprotein E in the sample. Related.

[0006] In certain embodiments, the methods provided herein comprise determining apolipoproteins in a sample. The purpose of the method is to determine the ApoE (ApoE) phenotype of a sample, the method comprising: (a) determining the ApoE phenotype of a sample; (b) purifying ApoE in said sample and isolating one or more ApoEs from said sample; (c) mass spectrometry to identify the stepwise sequence of ApoE ions. detecting ions derived from step (b), wherein in step (c) The identity of the emitted ions determines the ApoE alleles present in the sample.

[0007] In some embodiments, the purification steps provided herein include liquid chromatography. In some embodiments, the liquid chromatography comprises high performance liquid chromatography. Includes fee (HPLC).

[0008] In some embodiments, the ApoE in the sample is digested. In some embodiments, ApoE is digested with trypsin. In some embodiments, the ApoE is microwaved. It is digested using microwave technology.

[0009] In some embodiments, the purification steps provided herein involve solid phase extraction (SPE). include.

[0010] In some embodiments, the ionization comprises electrospray ionization (ESI). In some embodiments, ionizing comprises ionizing in a positive mode. In some embodiments, ionization includes ionizing in a negative mode. .

[0011] In some embodiments, the methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled.

[0012] In some embodiments, the phenotype determined by the methods provided herein is Ap In some embodiments, the phenotype is ApoE2 / ApoE In some embodiments, the phenotype is ApoE2 / ApoE4. In some embodiments, the phenotype is ApoE3 / ApoE3. In some embodiments, the phenotype is ApoE3 / ApoE4. oE4 / ApoE4.

[0013] In some embodiments, ApoE2 / ApoE2 has a mass / In some embodiments, the Apo E2 / ApoE2 is a precursor ion with a mass / charge ratio of 612.19±0.5. In some embodiments, ApoE2 / ApoE2 is determined by a mass / charge ratio selected from the group consisting of 835.93±0.5 and 835.93±0.5; In some embodiments, the ApoE2 / Ap fragment ions are determined by the presence of the fragment ions. oE2 has mass / charge ratios of 665.72±0.5 and 835.93±0.5. In some embodiments, the ApoE2 / Ap fragment ions are determined by the presence of the fragment ions. oE2 is a quality selected from the group consisting of 866.99±0.5 and 982.08±0.5 The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In this study, the ApoE2 / ApoE2 ratio was 866.99±0.5 and 982.08±0.5. The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In the meantime, ApoE2 / ApoE2 were 665.72±0.5, 835.93±0.5, and 86 a mass / charge ratio selected from the group consisting of 6.99±0.5, and 982.08±0.5 In some embodiments, the Apo E2 / ApoE2: 665.72±0.5, 835.93±0.5, 866.99±0 0.5, and the presence of fragment ions with mass / charge ratios of 982.08 ± 0.5. is determined by.

[0014] In some embodiments, ApoE2 / ApoE3 has a mass / In some embodiments, the Apo E2 / ApoE3 is a precursor ion with a mass / charge ratio of 612.19±0.5. In some embodiments, ApoE2 / ApoE3 is determined by It is determined by the presence of precursor ions with a mass / charge ratio of ±0.5. In terms of morphology, ApoE2 / ApoE3 were 665.72±0.5 and 835.93±0.5 determined by the presence of fragment ions having a mass / charge ratio selected from the group consisting of In some embodiments, ApoE2 / ApoE3 is 665.72±0.5 and and the presence of fragment ions with a mass / charge ratio of 835.93±0.5. In some embodiments, ApoE2 / ApoE3 is 866.99±0.5 and and a fragment having a mass / charge ratio selected from the group consisting of 982.08±0.5 In some embodiments, ApoE2 / ApoE3 is determined by the presence of Fragments with mass / charge ratios of 866.99±0.5 and 982.08±0.5 In some embodiments, ApoE2 / ApoE3 is determined by the presence of Mass / charge ratios selected from the group consisting of 374.42±0.5 and 502.55±0.5 In some embodiments, the Ap ApoE2 / ApoE3 have mass / charge ratios of 374.42±0.5 and 502.55±0.5. In some embodiments, the Ap oE2 / ApoE3 were 374.42±0.5, 502.55±0.5, and 665.72± 0.5, 835.93±0.5, 866.99±0.5, and 982.08±0.5 and the presence of fragment ions having a mass / charge ratio selected from the group consisting of: In some embodiments, ApoE2 / ApoE3 is 374.42±0.5, 50 2.55±0.5, 665.72±0.5, 835.93±0.5, 866.99±0. 5, and the presence of fragment ions with mass / charge ratios of 982.08 ± 0.5. is determined.

[0015] In some embodiments, ApoE2 / ApoE4 has a mass / In some embodiments, the Apo E2 / ApoE4 has a precursor ion with a mass / charge ratio of 612.19±0.5. In some embodiments, ApoE2 / ApoE4 is determined by It is determined by the presence of precursor ions with a mass / charge ratio of ±0.5. In the form ApoE2 / ApoE4 has a mass / charge ratio of 503.56±0.5 In some embodiments, the ApoE2 / ApoE4 is a mass / electron ratio selected from the group consisting of 665.72±0.5 and 835.93±0.5 In some embodiments, the ratio of the charge ratios is determined by the presence of fragment ions. ApoE2 / ApoE4 had mass / voltage ratios of 665.72±0.5 and 835.93±0.5. In some embodiments, the ratio of the charge ratios is determined by the presence of fragment ions. ApoE2 / ApoE4 is a group consisting of 866.99±0.5 and 982.08±0.5 The presence of fragment ions having a mass / charge ratio selected from In some embodiments, ApoE2 / ApoE4 is 866.99±0.5 and 982. The presence of fragment ions with a mass / charge ratio of 0.8±0.5 is determined. In some embodiments, ApoE2 / ApoE4 is 374.42±0.5 and 502. The presence of fragment ions having a mass / charge ratio selected from the group consisting of 55±0.5. In some embodiments, ApoE2 / ApoE4 is determined by the presence of The presence of fragment ions with mass / charge ratios of 2.2±0.5 and 502.55±0.5 In some embodiments, ApoE2 / ApoE4 is determined by the presence of having a mass / charge ratio selected from the group consisting of 4±0.5 and 892.96±0.5 In some embodiments, the ApoE2 / A fragment ion is determined by the presence of the fragment ion. poE4 has a mass / charge ratio of 649.74±0.5 and 892.96±0.5 In some embodiments, the ApoE2 / A fragment ion is determined by the presence of the fragment ion. poE4: 374.42±0.5, 502.55±0.5, 649.74±0.5, 6 65.72±0.5, 835.93±0.5, 866.99±0.5, 892.96±0 0.5, and 982.08±0.5. In some embodiments, the ApoE2 / ApoE2 fragment is determined by the presence of a fragment ion. E4: 374.42±0.5, 502.55±0.5, 649.74±0.5, 665 0.72±0.5, 835.93±0.5, 866.99±0.5, 892.96±0.5 , and the presence of a fragment ion with a mass / charge ratio of 982.08±0.5. It is decided.

[0016] In some embodiments, ApoE3 / ApoE3 has a mass / In some embodiments, the Apo E3 / ApoE3 is a precursor ion with a mass / charge ratio of 475.05±0.5. In some embodiments, ApoE3 / ApoE3 is determined by a mass / charge ratio selected from the group consisting of 982.08±0.5 and 982.08±0.5; In some embodiments, the ApoE3 / Ap fragment ions are determined by the presence of the fragment ions. oE3 has a mass / charge ratio of 866.99±0.5 and 982.08±0.5. In some embodiments, the ApoE3 / Ap fragment ions are determined by the presence of the fragment ions. oE3 is a quality selected from the group consisting of 374.42±0.5 and 502.55±0.5 The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In this study, the ApoE3 / ApoE3 ratio was 374.42±0.5 and 502.55±0.5. The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In the meantime, ApoE3 / ApoE3 were 374.42±0.5, 502.55±0.5, and 86 a mass / charge ratio selected from the group consisting of 6.99±0.5, and 982.08±0.5 In some embodiments, the Apo E3 / ApoE3: 374.42±0.5, 502.55±0.5, 866.99±0 0.5, and the presence of fragment ions with mass / charge ratios of 982.08 ± 0.5. is determined by.

[0017] In some embodiments, ApoE3 / ApoE4 has a mass / In some embodiments, the Apo E3 / ApoE4 is a precursor ion with a mass / charge ratio of 475.05±0.5. In some embodiments, ApoE3 / ApoE4 is determined by 503.56 It is determined by the presence of precursor ions with a mass / charge ratio of ±0.5. In terms of morphology, ApoE3 / ApoE4 were 866.99±0.5 and 982.08±0.5 determined by the presence of fragment ions having a mass / charge ratio selected from the group consisting of In some embodiments, ApoE3 / ApoE4 is 866.99±0.5 and and the presence of fragment ions with a mass / charge ratio of 982.08±0.5. In some embodiments, ApoE3 / ApoE4 is 374.42±0.5 and and a fragment having a mass / charge ratio selected from the group consisting of 502.55±0.5 In some embodiments, ApoE3 / ApoE4 is determined by the presence of Fragments with mass / charge ratios of 374.42±0.5 and 502.55±0.5 In some embodiments, ApoE3 / ApoE4 is determined by the presence of Mass / charge ratios selected from the group consisting of 649.74±0.5 and 892.96±0.5 In some embodiments, the Ap ApoE3 / ApoE4 have mass / charge ratios of 649.74±0.5 and 892.96±0.5. In some embodiments, the Ap oE3 / ApoE4: 374.42±0.5, 502.55±0.5, 649.74± 0.5, 866.99±0.5, 892.96±0.5, and 982.08±0.5 and the presence of fragment ions having a mass / charge ratio selected from the group consisting of: In some embodiments, ApoE3 / ApoE4 is 374.42±0.5, 50 2.55±0.5, 649.74±0.5, 866.99±0.5, 892.96±0. 5, and the presence of fragment ions with mass / charge ratios of 982.08 ± 0.5. is determined.

[0018] In some embodiments, ApoE3 / ApoE4 has a mass / In some embodiments, the Apo E3 / ApoE4 has a precursor ion with a mass / charge ratio of 503.56±0.5. In some embodiments, ApoE3 / ApoE4 is determined by a mass / charge ratio selected from the group consisting of 502.55±0.5 and 502.55±0.5; In some embodiments, the ApoE3 / Ap fragment ions are determined by the presence of the fragment ions. oE4 has a mass / charge ratio of 374.42±0.5 and 502.55±0.5. In some embodiments, the ApoE3 / Ap fragment ions are determined by the presence of the fragment ions. oE4 has a quality selected from the group consisting of 649.74±0.5 and 892.96±0.5 The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In this study, the quality of ApoE3 / ApoE4 was 649.74±0.5 and 892.96±0.5. The presence of fragment ions having a mass / charge ratio of 0.01 to 0.02 is determined by the presence of fragment ions having a mass / charge ratio of 0.01 to 0.02. In the ApoE3 / ApoE4 range, the ratios were 374.42±0.5, 502.55±0.5, and 64. a mass / charge ratio selected from the group consisting of 9.74±0.5, and 892.96±0.5 In some embodiments, the Apo E3 / ApoE4: 374.42±0.5, 502.55±0.5, 649.74±0 0.5, and the presence of fragment ions with mass / charge ratios of 892.96 ± 0.5. is determined by.

[0019] In some embodiments, the presence of the ApoE4 allele is associated with a risk of developing Alzheimer's disease. In some embodiments, the presence of the ApoE4 / ApoE4 allele is indicative of an increased risk of HIV infection. Presence of cerebrospinal fluid suggests an increased risk of developing Alzheimer's disease.

[0020] In some embodiments, quantification of total ApoE is performed using a mass / charge ratio of 485.06±0.5. In some embodiments, the determination of total ApoE includes measuring precursor ions having a ratio. The amount is a mass / charge ratio selected from 489.51±0.5 and 588.64±0.5. The method includes measuring fragment ions having the following structure:

[0021] In certain embodiments, the limit of quantitation of the method is less than or equal to 10 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 5 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 4 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 3 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 2 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 1 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 0.5 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 0.2 ng / mL. In this embodiment, the limit of quantitation of the method is less than or equal to 0.1 ng / mL.

[0022] In some embodiments, the limit of detection 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 limit of detection 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, or equal to, 0.05 ng / mL. In some embodiments, the detection limit of the method is less than 0.01 ng / mL or less. is equal to.

[0023] In some embodiments, ApoE is underivatized prior to mass spectrometry. stomach.

[0024] In some embodiments, ApoE is derivatized prior to mass spectrometry. .

[0025] In certain embodiments, the sample is a bodily fluid. In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. The pull is saliva or urine.

[0026] In some embodiments, the method comprises adding a drug to the sample in an amount sufficient to deproteinize the sample. This may involve adding an agent to the sample.

[0027] As used herein, unless otherwise indicated, the singular forms "a," "an," and "the" are used. " includes plural meaning. Thus, for example, when we say "one protein," we mean multiple proteins. Protein molecules are also included.

[0028] As used herein, the term "purification" or "to purify" refers to the removal of a desired component from a sample. Purification does not mean the removal of all substances other than the analyte of interest. the concentration of the analyte of interest relative to other components in the sample that may interfere with the detection of the analyte of interest. A sample, as used herein, refers to a procedure that enriches the amount of one or more analytes that are present in a sample. is one or more interfering substances, e.g., selected AP by mass spectrometry Various techniques are available that allow for the removal of one or more substances that interfere with the detection of parent and daughter ions of oE. It is refined in stages.

[0029] As used herein, the term "test sample" refers to any sample that may contain ApoE. As used herein, the term "body fluid" means any fluid that can be isolated from the body of an individual. For example, "body fluids" includes blood, plasma, serum, bile, saliva, urine, tears, Examples include sweat.

[0030] As used herein, the term "derivatizing" refers to reacting two molecules to form a new Derivatization agents include isothiocyanate groups, dinitrofluoro groups, containing a phenyl group, a nitrophenoxycarbonyl group, and / or a phthalaldehyde group, etc. obtain.

[0031] As used herein, the term "chromatography" refers to the process of separating particles carried by a liquid or gas. When a chemical mixture flows around or above a stationary liquid or solid phase, chemical entities The term "separation" refers to the process of separation into components as a result of differential partitioning of

[0032] As used herein, the term "liquid chromatography" or "LC" refers to the When a fluid permeates uniformly through a column of a substance or through a capillary passage, It refers to the process by which one or more components of a fluid solution are selectively retarded. or between multiple stationary phases and the bulk liquid (i.e., mobile phase), where this fluid is in phase with the stationary phase It is caused by the distribution of components in a mixture as they move relative to one another. Examples of "-" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), High-performance liquid chromatography (HPLC), and high-turbulence liquid chromatography (HTLC).

[0033] As used herein, the term "high performance liquid chromatography" or "HPLC" means by applying force to the mobile phase under pressure through a stationary phase, typically a densely packed column. This means that the degree of separation is increased by the use of liquid chromatography.

[0034] As used herein, the term "high turbulence liquid chromatography" or "HTLC" means The basic principle for carrying out separation is the turbulent flow of the substance to be assayed through the column packing. HTLC refers to a form of chromatography that uses mass spectrometry. It has been applied to prepare samples containing two unnamed drugs prior to analysis by HPLC. For example, Zimmer et al., J. Chromatogr. A854:23-35 (1999) See U.S. Patent No. 5,968,367, which further describes HTLC; See also Nos. 5,919,368, 5,795,469, and 5,772,874. Those skilled in the art understand "turbulent flow." When a fluid flows slowly and smoothly, For example, the flow moving at a low flow rate through an HPLC column is called laminar flow. The flow is laminar. In laminar flow, the movement of particles in the fluid is orderly and the particles move in straight lines. At higher speeds, the inertial force of the water exceeds the frictional force of the fluid, resulting in turbulence. Fluids that do not come into contact with irregular boundaries are slowed down by friction or flow through uneven surfaces. When a fluid flows in a turbulent state, it "overtakes" the fluid that has changed direction due to the surface. The flow is swirling (or vortex-like), which makes it more "resistant" than if the flow were stratified. There are many references available to help determine whether a fluid flow is laminar or turbulent. (e.g. 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 Press s (2001)).

[0035] As used herein, the term "gas chromatography" or "GC" refers to the process of analyzing a sample. The mixture is vaporized and passed through a column containing a stationary phase composed of a liquid or particulate solid. The compound is injected into a flow of carrier gas (nitrogen or helium) that travels through the It refers to chromatography in which a compound is separated into its constituent compounds based on its affinity for the stationary phase. Taste.

[0036] As used herein, the term "large particle column" or "extraction column" refers to a column having a particle size of approximately 35 μm. In this context, the term "chromatography column" refers to a column containing particles with a mean particle size greater than m. When used herein, the term "about" means ±10%. It contains particles approximately 60 μm in diameter.

[0037] As used herein, the term "analytical column" refers to a column that allows the separation of materials in a sample from the column. for sufficient separation to allow determination of the presence, absence, or amount of analyte. means a chromatography column having a chromatographic plate. The system then extracts the retained material to obtain a purified sample for further analysis. Many of these come from "extraction columns" which have the general purpose of separating or extracting substances from others. When used in this context, the term "about" means ±10%. In a preferred embodiment, the analytical column contains particles approximately 4 μm in diameter.

[0038] As used herein, the term "online automated method" or "online extraction" refers to The terms "online" or "inline" as used herein mean a system that requires no operator intervention. In contrast, the term "offline" refers to a procedure that is performed without the need for a When used in this context, it means a procedure that requires manual intervention by an operator. If the sample is precipitated and then the supernatant is manually loaded into the autosampler, The settling and loading steps are off-line from the subsequent steps. In some embodiments, one or more steps may be performed in an online, automated manner.

[0039] As used herein, the term "mass spectrometry" or "MS" refers to the method of analyzing a compound. MS refers to analytical techniques that identify ions by their mass-to-charge ratio, or 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) ionizing the charged compound. The method generally involves determining the molecular weight of the compound and calculating the mass-to-charge ratio. Ionization and detection may be by any suitable means. A "mass spectrometer" is an ionization device. and an ion detector. Generally, one or more molecules of interest are ionized. The ions are then introduced into a mass spectrometric instrument, where magnetic and electric fields The combination of these causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). For example, "Mass Spectrometry From Surface No. 6,204,500, entitled "Methods and Apparatus for The same paper, entitled "Abstract for Tandem Mass Spectrometry" No. 6,107,623, “DNA Diagnostics Based On Mass No. 6,268,144, entitled "Surface Spectrometry" -Enhanced Photolabile Attachment And Rel ease For Desorption And Detection Of Ana No. 6,124,137, entitled "Prostate Lytes"; Wright et al., Prostate Lytes Cancer and Prostatic Diseases, Vol. 2: 264–76 pp. (1999); and Merchant and Weinberger, Electro See phoresis, 21:1164-67 (2000).

[0040] As used herein, the term "operating in negative ion mode" refers to a mode in which negative ions are generated and detected. The term "operating in positive ion mode" means a mass spectrometry method in which: As used herein, it refers to a mass spectrometry method in which positive ions are generated and detected. do.

[0041] As used herein, the term "ionization" or "ionizing" refers to one or more The process of producing analyte ions with a net charge equal to 1 electron unit is called An anion is an ion that has a net negative charge of one or more electron units. whereas cations are ions with a net positive charge of one or more electron units. It is.

[0042] As used herein, the term "electron ionization method" or "EI method" refers to a gas phase The way in which the analyte of interest in the gas or vapor phase interacts with the electron flow The impact of electrons on the analyte produces analyte ions, which are then analyzed by mass spectrometry. It can be the subject of technology.

[0043] As used herein, the term "chemical ionization" or "CI" refers to a method using a reagent gas (e.g., The reaction mixture (e.g., ammonia) is bombarded with electrons and reacts with the reagent gas ions and analyte molecules. By "analyte ion" is meant the manner in which the analyte ion is formed.

[0044] As used herein, the term "fast atom bombardment" or "FAB" refers to a method for producing high-energy A beam of atoms (often Xe or Ar) bombards a non-volatile sample, The test sample is a viscous liquid. Body matrices, such as glycerol, thioglycerol, m-nitrobenzyl alcohol 18-crown-6-crown ether, 2-nitrophenyl octyl ether, It dissolves in methylpropanol, diethanolamine, and triethanolamine. Selection of a suitable matrix for a sample is an empirical process.

[0045] As used herein, the term "matrix-assisted laser desorption ionization" or "MA" refers to a method for detecting a nucleophilic substance. "LDI" refers to various processes including photoionization, protonation, deprotonation, and cluster decay. The non-volatile sample is then ionized by a suitable ionization pathway, which desorbs and ionizes the analytes in the sample. In the case of MALDI, the sample is exposed to laser radiation that causes the analysis It is mixed with an energy absorbing matrix that promotes the desorption of the substance molecules.

[0046] As used herein, the term "surface-enhanced laser desorption ionization" or "SELDI" " refers to various ionization processes including photoionization, protonation, deprotonation, and cluster decay. The ionization pathway allows a non-volatile sample to desorb and ionize the analytes in the sample. In SELDI, the sample is exposed to one or more laser beams. Typically bound to a surface that preferentially retains multiple analytes of interest. Similar to MALDI. Additionally, this process may also utilize an energy absorbing material to facilitate ionization.

[0047] As used herein, the term "electrospray ionization" or "ESI" refers to A solution is passed along a short length of capillary tube, and a positive or negative high voltage is attached to the end of the capillary tube. This refers to the way in which an electric potential is applied. When the solution reaches the end of the tube, it is vaporized (atomized) and Within the solvent vapor, a jet or spray of very small droplets of the solution forms. The mist passes through a slightly heated evaporation chamber to prevent condensation and evaporate the solvent. As the droplets become smaller, the surface charge density increases, and eventually the droplets move spontaneously between the same charge. The natural repulsion causes the ejection of ions as well as neutral molecules.

[0048] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to ES It refers to a mass spectrometry technique similar to I, but APCI occurs in a plasma at atmospheric pressure. Ions are produced by ion-molecule reactions. The plasma is generated between the spray capillary and the counter electrode. The ions are then pumped through a series of differentially pumped skimmers. It is typically extracted into the mass spectrometer by the use of a dry and preheated N2 Countercurrent gas flow can be used to improve solvent removal. In some cases, gas phase ionization in APCI can be more effective than ESI.

[0049] The term "atmospheric pressure photoionization" or "APPI" as used herein refers to a method for detecting molecular The mechanism for photoionizing M is photon absorption and electron emission to form the molecular ion M+. This refers to the form of analytical method. The photon energy is generally just above the ionization potential, so The molecular ion is less likely to dissociate, often requiring chromatography. It is believed that samples can be analyzed without the need for a separate sample, thus saving considerable time and expense. In the presence of water vapor or a protic solvent, the molecular ion can be converted to MH+ by removing H. This tends to occur when M has a high proton affinity. This does not affect the accuracy of the quantification, since the sum of M+ and MH+ is constant. Drug compounds in polar solvents are usually observed as MH+, while nonpolar compounds, e.g., naphthyl Taren or testosterone usually take the M+ form. Robb, DB, Cove y, TR and Bruins, AP (2000): See, e.g., Robb et al., Atm ospheric pressure photoionization:An ion ization method for liquid chromatography -mass spectrometry. Anal. Chem. Vol. 72(Issue 15):3 See pages 653-3659.

[0050] As used herein, the term "inductively coupled plasma" or "ICP" refers to At a sufficiently high temperature that the elements are atomized and ionized, the sample is partially ionized. It refers to the way in which the electrons interact with the ionized gas.

[0051] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is ionized. This refers to a method in which a strong electric field is used to generate analyte ions, which are placed on a surface. do.

[0052] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or means that the analyte is introduced into the gas phase.

[0053] As used herein, the terms "limit of quantification" and "quantitation limit" are used interchangeably. "Limit of quantitation," or "LOQ," means the limit at which a measurement is quantitatively meaningful. The analyte response at this LOQ is distinguishable and unique. It is unique and has a precision of 20% and a reproducibility of 80% to 120% accuracy.

[0054] As used herein, the term "limit of detection" or "LOD" refers to the limit at which a measurement is made. The LOD is the point at which the uncertainty in the concentration is greater than the uncertainty in the concentration. is arbitrarily defined as

[0055] As used herein, the "amount" of ApoE in a body fluid sample refers to the amount of ApoE detected in a volume of body fluid. Generally, the absolute value reflecting the mass of ApoE available is meant. However, the amount may differ depending on the ApoE For example, the amount of ApoE in body fluids is usually It can be an amount that is above or below the control or normal level of ApoE present. .

[0056] The term "about" as used herein in connection with quantitative measurements, excluding ion mass measurements, means The indicated value means ±10%. Mass spectrometry instruments calculate the mass of a given analyte. The term "about" refers to the mass of an ion or the mass of an ion. In the context of the charge / charge ratio, this means ±0.5 atomic mass units.

[0057] The above summary of the invention is not limiting, and other features and advantages of the invention are set forth below. This will be apparent from the detailed description and from the claims. [Brief explanation of the drawings]

[0058] [Figure 1-1] FIG. 1 shows an example chromatogram of the ApoE2 / E2 phenotype, which has a frequency of about 0.2%. [Figure 1-2] FIG. 1 shows an example chromatogram of the ApoE2 / E2 phenotype, which has a frequency of about 0.2%. [Figure 2-1] FIG. 1 shows an example chromatogram of the ApoE2 / E3 phenotype, which has a frequency of about 9.4%. [Figure 2-2] FIG. 1 shows an example chromatogram of the ApoE2 / E3 phenotype, which has a frequency of about 9.4%. [Figure 3-1] FIG. 1 shows an example chromatogram of the ApoE2 / E4 phenotype, which has a frequency of about 2.2%. [Figure 3-2] FIG. 1 shows an example chromatogram of the ApoE2 / E4 phenotype, which has a frequency of about 2.2%. [Figure 4-1] FIG. 1 shows an example chromatogram of the ApoE3 / E3 phenotype, which has a frequency of about 66%. [Figure 4-2] FIG. 1 shows an example chromatogram of the ApoE3 / E3 phenotype, which has a frequency of about 66%. [Figure 5-1] FIG. 1 shows an example chromatogram of the ApoE3 / E4 phenotype, which has a frequency of approximately 20%. [Figure 5-2] FIG. 1 shows an example chromatogram of the ApoE3 / E4 phenotype, which has a frequency of approximately 20%. [Figure 6-1] FIG. 1 shows an example chromatogram of the ApoE4 / E4 phenotype, which has a frequency of about 2.5%. [Figure 6-2] FIG. 1 shows an example chromatogram of the ApoE4 / E4 phenotype, which has a frequency of about 2.5%. [Figure 7] FIG. 1 shows ApoE allele frequencies based on 319 individual serum samples as determined by LC-MS / MS. [Figure 8] Figure 1 shows the contribution of each Alzheimer's disease biomarker to the risk assessment model. The formula for calculating the linear predictor (score) for MCI or Alzheimer's disease is: score = 2.8336 - 9.9026 x ratio + 0.7358 x ApoE4 - 0.2183 x total ApoE, where Aβ42 (pg / mL) / Aβ40 (pg / mL) ratio; ApoE4 allele count; total ApoE (μg / mL). Risk is categorized into three groups: low risk; average risk; and high risk. [Figure 9] FIG. 1 shows plots of disease probability against the Aβ42 / 40 ratio model by allele number. [Figure 10]FIG. 1 shows plots of disease risk for the Aβ42 / 40 ratio + total ApoE model by ApoE4 allele number. [Figure 11] FIG. 1 shows risk assessment scores versus number of ApoE4 alleles. [Figure 12] FIG. 1 shows a logistic regression analysis model for ADMark. [Figure 13] Figure 1 shows a graphical representation of ApoE isotype phenotyping by mass spectrometry. ApoE2 / E2 phenotype is determined by detecting unique E2 associated ions. DETAILED DESCRIPTION OF THE INVENTION

[0059] Apolipoprotein E (ApoE) is a potential therapeutic target for late-onset Alzheimer's disease (AD). The human APOE gene is a well-defined genetic risk factor for glaucoma. ε2 / ε2, ε3, and ε4, resulting in six different phenotypes: ε2 / ε2, This results in ε2 / ε3, ε3 / ε3, ε2 / ε4, ε3 / ε4, and ε4 / ε4. Approximately half of individuals (compared to 14% in the general population) carry the ε4 allele, the majority of whom Heterozygotes (ε3 / ε4). The number of inherited ε4 alleles varies depending on the number of ε2 or ε3 alleles. Associated with both an increased disease risk and a decreased mean age of onset compared with genetic inheritance The differences between the three ApoE isoforms are due to their structure and therefore the protein and various Based on two amino acids that affect the interaction and binding with lipids and β-amyloid (Aβ) ApoE and Aβ may coexist in the brain, and thus their complementary roles in AD may be important. Circulating plasma and CSF ApoE levels are associated with ApoE-mediated vasoconstriction. Recently, CSF Apo-E has been shown to be a potential biomarker for Apo-E. Increased levels of α-2 or α-E3 may represent a protective response to injury in AD. In addition to its effect on amyloid clearance, tau and amyloid deposition It may have neuroprotective effects by independently reducing neuronal injury. Decreased cholesterol levels are also associated with multiple sclerosis and other neurodegenerative diseases that affect brain lipid metabolism. It is possible that

[0060] In certain embodiments, the methods provided herein comprise determining apolipoproteins in a sample. The purpose of the method is to determine the ApoE (ApoE) phenotype of a sample, the method comprising: (a) determining the ApoE phenotype of a sample; (b) purifying ApoE in said sample to obtain a purified ApoE; (c) generating one or more ions; and (c) determining the step by mass spectrometry. and detecting ions derived from group (b), wherein the A present in the sample The poE allele is determined from the identity of the ion detected in step (c). .

[0061] In some embodiments, the purification steps provided herein include liquid chromatography. In some embodiments, the liquid chromatography comprises high performance liquid chromatography. Includes fee (HPLC).

[0062] In some embodiments, the purification steps provided herein involve solid phase extraction (SPE). include.

[0063] In some embodiments, the ionization comprises electrospray ionization (ESI). In some embodiments, ionizing comprises ionizing in a positive mode. In some embodiments, ionization includes ionizing in a negative mode. .

[0064] In some embodiments, the methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled.

[0065] In some embodiments, the phenotype determined by the methods provided herein is Ap In some embodiments, the phenotype is ApoE2 / ApoE In some embodiments, the phenotype is ApoE2 / ApoE4. In some embodiments, the phenotype is ApoE3 / ApoE3. In some embodiments, the phenotype is ApoE3 / ApoE4. oE4 / ApoE4.

[0066] In some embodiments, the presence of the ApoE4 allele is associated with a risk of developing Alzheimer's disease. In some embodiments, the presence of the ApoE4 / ApoE4 allele is indicative of an increased risk of HIV infection. Presence of cerebrospinal fluid suggests an increased risk of developing Alzheimer's disease.

[0067] In certain embodiments, the limit of quantitation of the method is less than or equal to 10 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 5 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 4 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 3 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 2 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 1 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 0.5 ng / mL. In embodiments, the limit of quantitation of the method is less than or equal to 0.2 ng / mL. In this embodiment, the limit of quantitation of the method is less than or equal to 0.1 ng / mL.

[0068] In some embodiments, the limit of detection 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 limit of detection 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, or equal to, 0.05 ng / mL. In some embodiments, the detection limit of the method is less than 0.01 ng / mL or less. is equal to.

[0069] In some embodiments, ApoE is underivatized prior to mass spectrometry. stomach.

[0070] In some embodiments, ApoE is derivatized prior to mass spectrometry. .

[0071] In certain embodiments, the sample is a bodily fluid. In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. The pull is saliva or urine.

[0072] In some embodiments, the method comprises administering the agent in an amount sufficient to deplete the sample of proteins. This may include adding the compound to the sample.

[0073] Suitable test samples include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample; i.e., any biological sample. Samples obtained from biological sources, such as animals, cell cultures, organ cultures, etc. In a preferred embodiment, the sample is obtained from a mammal, such as a dog, cat, horse, etc. Particularly preferred mammals are primates, most preferably humans, male or female. Preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or Examples of such samples include tissue samples from patients; i.e., A living organism that presents itself in a clinical setting to diagnose, predict, or treat a disease or condition. The test sample is preferably obtained from a patient, e.g., a human, male or female. For example, serum.

[0074] Sample Preparation for Mass Spectrometry ApoE is more abundant than other components (e.g., proteins) in the sample. Methods that can be used to achieve this include, for example, filtration, centrifugation, thin layer deposition, and the like. Chromatography (TLC), electrophoresis including capillary electrophoresis, immunoaffinity affinity separation methods, including affinity separation methods; extraction methods, including ethyl acetate extraction methods and methanol extraction methods; and chaotropic agents, or any combination of the above.

[0075] Protein precipitation is one preferred method of preparing test samples. Protein purification methods are well known in the art and are described, for example, in Polson et al., Journal of al of Chromatography B, Vol. 785: 263-275 (20 (2003) describes protein precipitation techniques suitable for use in the present method. Protein precipitation removes most of the protein from the sample, leaving ApoE in the supernatant. The sample is centrifuged to separate the liquid supernatant from the precipitated proteins. The resulting supernatant can then be subjected to liquid chromatography and subsequent mass spectrometry. In certain embodiments, protein precipitation methods, e.g., acetylation, may be applied to chromatographic analysis. Using methods such as acetonitrile protein precipitation, HPLC and mass spectrometry can be performed. High turbulence liquid chromatography (HTLC) or other online extraction should be performed before Thus, in such an embodiment, the method comprises: (1) determining the target sample; (2) performing protein precipitation of the sample; and (3) performing on-line extraction or high-turbulence liquid chromatography. The supernatant was directly loaded onto an HPLC-mass spectrometer without the use of high-temperature chromatography (HTLC). It is related to doing.

[0076] In some preferred embodiments, HPLC is used alone or in combination with one or more purification steps. In combination with the method, it can be used to purify ApoE prior to mass spectrometry. In such an embodiment, the sample is extracted by an HPLC extraction cartridge that captures the analyte. The resulting mixture is extracted using a HPLC column, then eluted and chromatographed on a second HPLC column. The chromatographic method can be carried out by ionizing the chromatographically isolated ions, or by eluting the chromatographically isolated ions onto an analytical HPLC column before ionization. The steps involved in the chromatographic procedure can be linked in an automated manner, so that the purification of the analyte can be achieved. The need for operator involvement during this period can be minimized. This results in time and cost savings and eliminates the opportunity for operator error. can be removed.

[0077] For example, turbulence caused by HTLC columns and methods can enhance mass transfer rates. HTLC columns contain rigid particles, which are believed to improve separation properties. Components are separated by high chromatographic flow rates through a packed column. By using a high flow rate (e.g., 3-5 mL / min), turbulence is generated in the column, and the stationary phase and This results in a near complete interaction between the analytes of interest. As a result, high molecular weight species are not retained under turbulent flow conditions and therefore are associated with the biofluid matrix. The accumulation of linked macromolecules is avoided. The combined HTLC method reduces the need for lengthy sample preparation and significantly increases Such methods offer superior analytical performance to laminar flow (HPLC) chromatography. HTLC also enables direct injection of biological samples (plasma, urine, etc.) When injected directly, denatured proteins and other biological debris rapidly pass through the separation column. This blocking occurs quickly, making it difficult to achieve with conventional chromatography. is less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, preferably 0 It also allows for very small sample volumes of 0.1 mL.

[0078] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry include: See, for example, Zimmer et al., J. Chromatogr. A854, Vol. :23-35 (1999); U.S. Patent No. 5,968,367; U.S. Patent 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 subjected to a tandem chromatography as described above prior to loading onto the HTLC column. In an alternative preferred embodiment, the sample is subjected to protein precipitation. The HTLC extraction column is preferably a column that can be loaded directly onto the HTLC without further processing. In various embodiments, one or more steps of the method include: It may be performed in an online, automated manner. For example, in one embodiment, steps (i) to (v) are carried out in an online, automated manner. The step of detecting is performed online after steps (i) to (v).

[0079] Liquid chromatography (LC), including high performance liquid chromatography (HPLC), Traditional HPLC analysis is based on a relatively slow laminar flow technique. The laminar flow of the column relies on the column packing material, which is the basis for separating the analytes of interest from the sample. Those skilled in the art understand that separation in such columns is a diffusion process. HPLC has been successfully applied to the separation of compounds in biological samples, but significant amounts of Sample preparation is required before separation and subsequent analysis by mass spectrometry (MS). Furthermore, most HPLC systems are not compatible with mass spectrometry, making the technique labor-intensive. The equipment is not being used to its full potential, with one HPLC system being used for one MS instrument. As a result, it takes a long time to perform multiple assays. Need.

[0080] Regarding the use of HPLC for sample removal prior to mass spectrometry analysis, Various methods have been described, see, e.g., Taylor et al., Therapeutic Drugs. ug Monitoring, 22:608-12 (2000); and Salm et al., Clin. Therapeutics, Vol. 22, Suppl. B: B71-B85 (2 000).

[0081] Those skilled in the art will be able to select suitable HPLC equipment and columns for use with ApoE. A chromatographic column is a medium (i.e., a column) that facilitates the separation (i.e., fractionation) of chemical moieties. The medium generally includes fine particles. The particles are: It has a binding surface that interacts with various chemical moieties to facilitate separation of the chemical moieties. Suitable bonding surfaces are hydrophobic bonding surfaces, such as alkyl bonding surfaces. is a C-4, C-8, C-12, or C-18 bonded alkyl group, preferably C-18 bonded The chromatographic column may include an injection port for receiving a sample and a separation port. An outlet port is provided for discharging the effluent containing the separated sample. is when a sample (or pre-purified sample) is applied to the column at the injection port. The solution is eluted with a solvent or solvent mixture and discharged through the discharge port. The column can be selected to elute the analyte of interest. The analysis can be performed using gradient, isocratic, or polymorphic (i.e., mixed) modes. During chromatography, separation of materials can be affected by variables such as solvents. Selection of the eluent (also known as the "mobile phase"), elution mode, gradient conditions, temperature, etc. be affected.

[0082] In certain embodiments, the analyte is selected from those in which the analyte of interest is reversibly retained by the column packing material. The sample is then loaded onto the column under conditions where the sample is retained while one or more other substances are not retained. In such an embodiment, the first mobile phase conditions may be: The analyte of interest can be configured to be retained by the column, and the second mobile phase The conditions are then such that the unretained substances are washed out and the retained substances are removed from the column. Alternatively, the analyte may be one or more other substances. Samples are analyzed under mobile phase conditions such that the analytes of interest elute at different rates compared to the Such a procedure may involve the application of one or more of the samples to a column. the possibility of enriching the amount of one or more analytes of interest relative to a number of other components There is.

[0083] In one preferred embodiment, the HTLC is a hydrophobic column chromatography system. In certain preferred embodiments, Cohesive Tech nologies TurboFlow Cyclone P® Polymer The base column (particle size 60 μm, column dimensions 50 × 1.0 mm, pore size 100 Å) was used. In a related preferred embodiment, Phe is used, which has a hydrophilic end-capping. Synergi Polar-RP® Ether from nomenex Inc. Analytical column for bonded phenyl (particle size 4 μm, column dimensions 150 × 2.0 mm, pore size In certain preferred embodiments, HTLC and HPLC are performed using a mobile phase of The analysis is carried out using HPLC-grade ultrapure water and 100% methanol.

[0084] Careful valve selection and connector piping eliminates the need for any manual steps. Two or more chromatographic columns are used so that material passes from one column to the next. In a preferred embodiment, the valve selection and piping The work is managed by a computer that is pre-programmed to carry out the necessary steps. Most preferably, the chromatography system also includes a detector system, e.g. The system is connected to the S system in such an online manner. Simply place the tray of samples into the autosampler and the computer will do the rest. - performed under controlled conditions, so that the purification and analysis of all selected samples is complete .

[0085] In certain preferred embodiments, the ApoE or fragment thereof in the sample is ionized. In a particularly preferred embodiment, the chromatography is carried out by gas chromatography. It's not topography.

[0086] Detection and quantification by mass spectrometry In various embodiments, ApoE or a fragment thereof may be any polypeptide known to those of skill in the art. Mass spectrometry can be used to analyze fractionated samples. and producing charged molecules for further analysis. For example, ionization of a sample can be performed using electron ionization, chemical ionization, or the like. ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (AP CI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid Laser desorption ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization Ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption Ionization (SELDI), Inductively Coupled Plasma (ICP), and Particle Beam Ionization Those skilled in the art will appreciate that the choice of ionization method depends on the analyte being measured, the type of sample, and the type of ionization method being used. This can be determined based on the type of detector, the choice of positive mode or negative mode, etc. I understand.

[0087] In a preferred embodiment, ApoE or a fragment thereof is Ionization is performed by heated electrospray ionization (HESI) in the active mode. In an alternative embodiment, ApoE or a fragment thereof is expressed in a positive or negative manner. Electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in the ionization mode ) and ionize it.

[0088] After the sample is ionized, the resulting positively or negatively charged ions are The ions may be analyzed to determine their mass-to-charge ratio. Suitable analyzers for this purpose include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Ions are detected using several detection modes. For example, selected ions can be detected, i.e., in selective ion monitoring mode. Ions can be detected using a scanning inversion (SIM) or alternatively, the ions can be detected using a scanning inversion (SIM) mode. For example, using multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio can be determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" device, the A force proportional to the applied DC potential, the amplitude of the RF signal, and the mass / charge ratio acts on the oscillatory The voltage and amplitude are applied to ions in a radio frequency field. Selected so that only on ions travel the length of the quadrupole while all other ions are missed. Therefore, quadrupole instruments can be used to "filter" ions injected into the instrument. It can act as both a "mass detector" and a "mass detector."

[0089] By utilizing "tandem mass spectrometry" or "MS / MS", the resolution of MS technology This technique allows for the enhancement of the precursor ions (parent ions) generated from the target molecules. The precursor ions (also called ions) can be filtered in the MS instrument, and is then fragmented to produce a single molecule that is then subjected to analysis in a second MS step. Or, multiple fragment ions (also called daughter ions or product ions) result. By carefully selecting precursor ions, only ions produced by specific analytes are identified. They pass through a fragmentation chamber where they collide with atoms of an inert gas to form fragment ions. Both precursor ions and fragment ions are generated by ionization / fragmentation. MS / MS technology is an extremely powerful analytical tool because it reproducibly produces nucleotides under a set of defined conditions that catalyze their synthesis. For example, a combination of filtering / fragmentation can provide analytical tools to identify interfering substances. and can be used to remove ions, particularly in complex samples, such as biological samples. It can be useful.

[0090] Mass spectrometers generally perform ion scanning; that is, scanning over a predetermined range (e.g., 100 ~1000 amu), the relative abundance of each ion with a specific mass / charge is calculated using The results of the analyte assay, i.e., mass spectra, are provided to the user. can be correlated with the amount of analyte in the original sample by numerous methods known in the art. For example, provided that sampling and analytical parameters are carefully controlled, The relative abundance of a given ion can be calculated by converting the relative abundance to the absolute amount of the original molecule. Alternatively, molecular standards can be run alongside the samples. A standard curve is then constructed based on the ions generated from the standards. Using a standard curve, the relative abundance of a given ion is converted to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard may be used as a standard for calculating the amount of ApoE. Methods for generating and using such standard curves are well known in the art. It is well known in the art and one of skill in the art has the ability to select an appropriate internal standard. The isotope of ApoE can be used as an internal standard. The amount of the ion is then compared to the amount of the original molecule. Numerous other methods for associating with are well known to those skilled in the art.

[0091] One or more steps of the method may be performed using automated machinery. In embodiments, one or more purification steps are performed online, and more preferably In most cases, all of the purification and mass spectrometry steps are performed online. obtain.

[0092] In certain embodiments, such as when precursor ions are isolated for further fragmentation, In MS / MS and other methods, collision activation dissociation is used. It is often used to generate fragment ions for further detection. In D, precursor ions gain energy through collisions with inert gas, but then They fragment through a process called "unimolecular decomposition." The increase in vibrational energy Due to this, sufficient energy is introduced into the precursor ions so that certain bonds within the ions can be broken. It must be accumulated within the

[0093] In a particularly preferred embodiment, ApoE is detected and analyzed using MS / MS as follows: The sample is processed by liquid chromatography, preferably HPLC. The liquid solvent flow from the chromatographic column is then passed through the MS / MS analyzer. The solvent / analyte mixture enters the heated nebulizer interface and is then The analyte is converted to a vapor in a heated tube at the interface. The ions, e.g., precursor ions, pass through an aperture in the device and are ionized. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters. ions (i.e., "precursors" and "fragments") based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is the collision cell where ions are separated. The first quadrupole (Q1) of the mass spectrometer detects the mass-to-charge ratio of ApoE. The charge ratio is used to select the molecule. The precursor ion with the correct mass / charge ratio for ApoE is , can be introduced into the collision chamber (Q2), while other desired species with different mass / charge ratios can be introduced into the collision chamber (Q3). Any unwanted ions collide with the sides of the quadrupole and are removed. The argon atoms collide with neutral argon gas molecules and fragment. This process is called collisional activation. The fragment ions are introduced into quadrupole 3 (Q3). The ApoE fragment ions were selected while the other ions were be removed.

[0094] The method is an MS / MS method performed in either positive or negative ion mode. Using standard methods well known in the art, one skilled in the art can determine the quadrupole 3 (Q3 ) one or more fragments of a specific ApoE precursor ion available for selection in It has the ability to distinguish between ions.

[0095] When the precursor ion of ApoE contains an alcohol or amine group, the precursor ion Fragment ions corresponding to dehydration or deamination are formed, respectively. In the case of precursor ions containing hydroxyl groups, such fragment ions formed by dehydration are It is caused by the loss of one or more water molecules from the precursor ion (i.e., the precursor ion and the The mass-to-charge ratio difference between the fragment ions is about 18 for the loss of one water molecule, (For example, the loss of two water molecules is about 36.) For precursor ions containing amine groups, the loss of Such fragment ions formed by amines are composed of one or more ammonia molecules. caused by the loss of mass (i.e., the mass pairing between precursor ions and fragment ions) The difference in the ratio of charges is about 17 for the loss of one ammonia molecule, or 2 ammonia molecules. (The loss of about 34 units, etc.) Similarly, The precursor ions, including the cations, are usually one or more water molecules and / or one or more ammonia molecules. Form fragment ions corresponding to the loss of an amino acid molecule (i.e., precursor ion and fragment ion) The difference in mass-to-charge ratio between the cations is due to the loss of one water molecule and the addition of one ammonia molecule. (The loss of about 35% is related to the dehydration or deamination of the precursor ion.) Fragment ions are not unique fragment ions for a particular analyte. In a preferred embodiment of the present invention, MS / MS is carried out to identify at least one fragment of ApoE. The precursor ion is detected as a precursor ion with only one or more water molecules lost. and / or the loss of one or more ammonia molecules. This will be implemented so that

[0096] When an ion collides with the detector, it emits a pulse of electrons that is converted into a digital signal. The acquired data is plotted as the collected ion counts versus time. The resulting mass chromatogram is then transmitted to a computer that processes the data. The area under the peak corresponding to a particular ion, or The amplitude of such peaks is measured, and the area or amplitude is correlated with the amount of analyte of interest. In certain embodiments, fragment ions and / or precursor ions are The area under the curve or peak amplitude is measured to determine the amount of ApoE. To achieve this, a calibration standard curve based on peaks derived from one or more ions of an internal molecular standard is used. Using this, the relative abundance of a given ion can be converted to the absolute amount of the original analyte.

[0097] The following examples serve to illustrate the invention. These examples do not limit the scope of the present method. There is no intention to impose any restrictions on [Example]

[0098] Example 1: Determining ApoE Phenotype by Mass Spectrometry Reagent Summary: Table 1

[0099] [Table 1]

[0100] CSF apolipoprotein E (ApoE) isoforms were detected by LC-MS / MS assay. Identifying the genome will reveal three distinct isoforms of ApoE, which will then lead to phenotypic It can be used to predict the apolipoprotein E protein (ApoE2, Apo There are three alleles encoding ApoE3, ApoE4, and ApoE5, which are expressed codominantly. Six unique phenotypes are present: ApoE2 / E2, ApoE2 / E3, ApoE2 / E4, This results in ApoE3 / E3, ApoE3 / E4, and ApoE4 / E4.

[0101] To determine each ApoE phenotype, trypsin digestion of the protein was performed and solidified. Apo peptides are used as surrogates to distinguish each protein isoform. Unique peptides exist for both E2 and ApoE4 isoforms The ApoE3 isoform is a common isoform between the ApoE2 and ApoE3 isoforms. The peptides that correspond to the ApoE3 and ApoE4 isoforms, as well as the common peptides between the ApoE3 and ApoE4 isoforms, were identified. The internal standards for each isoform are determined using retention time reference points. It is spiked into each sample to serve as a control.

[0102] CSF ApoE samples were analyzed in triplicate using a Thermo Fisher Quantiva Thermo Aria Cohesive TLX-4 connected to a quadrupole mass spectrometer The data are analyzed using tandem mass spectrometry with high flow LC. The chromatograms were monitored by MRM and analyzed by Thermo Fisher LC Quasi-Quantum. Analyze using an data analysis software.

[0103] All mass-to-charge ratios (m / z) that identify the various ApoE alleles were analyzed using several This is shown in the figure and summarized in Table 2 below.

[0104] [Table 2]

[0105] Predicted values: apolipoprotein E in CSF: 2.84-7.24 μg / mL; in serum Apolipoprotein E: 20.07–101.68 μg / mL.

[0106] Five technical replicates for each quality control level were performed over five days, with low, medium, and low replicates performed each day. and were measured in order of increasing levels.

[0107] CSF low level quality control: 1.2μg / mL Apolipoprotein E: Average: 1.16~1.37 SD: 0.03 to 0.12 CV(%): 2.27~10.35% Recovery rate (%): 97.00-104.00%

[0108] CSF mid-level quality control: 3.0 μg / mL Apolipoprotein E: Average: 2.68~3.37 SD: 0.05 to 0.33 CV(%): 1.74~10.59% Recovery rate (%): 89.27~112.40%

[0109] CSF high-level quality control: 15.0 μg / mL Apolipoprotein E: Average: 13.38~16.54 SD: 0.40 to 1.65 CV(%): 4.71~11.36% Recovery rate (%): 89.20~110.29%

[0110] Accuracy: Patients with known APOE genotype (analysis method: Restriction Length Polymorphism Method (RLPM)) Twenty samples were analyzed by LC-MS / MS. ApoE phenotypes were then determined using known genes. As shown in Table 3 below, the genotype and phenotype were compared for each patient sample. There was 100% agreement between them.

[0111] [Table 3]

[0112] Freeze-thaw stability: The six spiked phenotypes were divided into four equal aliquots. The freeze-thaw analysis was performed by analyzing the aliquots for each phenotype. All were frozen at -90 to -60°C. The second to fourth aliquots were subjected to a single freeze-thaw cycle. For freezing, thaw at ambient temperature of 18-25°C and then freeze. The aliquot is thawed at ambient temperature between 18 and 25°C for two freeze-thaw cycles, and The fourth aliquot was then frozen at ambient temperature between 18 and 25°C for three freeze-thaw cycles. The mixture was thawed at 37°C and frozen.

[0113] Finally, all aliquots were thawed at ambient temperature between 18 and 25 °C and then analyzed in technical triplicate. The freeze-thaw analysis included data for three freeze-thaw cycles. The poE phenotype has acceptable stability for up to three freeze-thaw cycles. Table 4 :

[0114] [Table 4]

[0115] Stability of sample after extraction: On the same day as the baseline sample was extracted, The next day, the same sample was reinjected and analyzed against the baseline value. This assay yields enough sample for two injections. ApoE is expressed in CTCs. Post-extraction sample stability for at least 1 day at 2-8°C in the C stack of the GC / MS sampler Table 5:

[0116] [Table 5]

[0117] Room temperature stability: Samples are stable for up to 7 days at 18-25°C. Table 6:

[0118] [Table 6]

[0119] Refrigerated Stability: Samples are stable at 2-8°C for up to 7 days. Table 7:

[0120] [Table 7]

[0121] Freezing stability: Samples are stable at -30 to -10°C for at least 31 days. Table 8:

[0122] [Table 8]

[0123] Disturbance Test Pass / fail criteria: Differences due to potential interfering substances should be ≤2 SD, or 20% The CV is considered acceptable.

[0124] Interference due to hemolysis: Six patient pools were analyzed using hemoglobin (Sigma Cat. No. H7 379) and baseline, minor, moderate, and major hemolysis The interference was analyzed in triplicate. A 10 mg / mL hemoglobin solution was used to detect "large" interference. For medium and small scale interferences, a 10 mg / mL solution was used. They were diluted 1:10 and 1:20 with BS, respectively.

[0125] Any level of hemolysis is unacceptable due to possible contamination with apolipoprotein E from serum. Not permitted. Table 9:

[0126] [Table 9]

[0127] Lipidemia interference: Six patient pools were treated with Intralipid (Sigma catalog no. No. I141), and baseline, small, medium, and large lipemia The interference caused by the disease was analyzed in triplicate. ) was used for "large" disturbances. For medium and small disturbances, 1:5 solvent The solutions were diluted 1:10 and 1:20 with 10 mM PBS, respectively. Regarding the degree of lipemia in the F samples, all of them are acceptable. Table 10:

[0128] [Table 10]

[0129] Bilirubin interference: Six patient pools were analyzed using bilirubin (Sigma catalog no. B4126) and baseline, minor, moderate, and major jaundice The interference caused by 1 mg / mL bilirubin was analyzed in triplicate. A 1 mg / mL solution was used for medium and small scale interferences, and 10 mM P The CSF samples were diluted 1:10 and 1:20 with BS, respectively. ApoE was detected in the CSF samples at the same time as the jaundice level. Table 11:

[0130] [Table 11]

[0131] Ion suppression: Ten patient samples were extracted. Ten samples were run through an analytical column. The ApoE digest peptide mixture was injected post-column. When the ApoE internal standard eluted, the total ion chromatogram of ApoE ( If the TIC showed a ≥ 15% decrease in signal intensity, the assay was The TIC of the digested peptides of ApoE was determined to be The TIC signal intensity was constant and showed no suppression within the gradient. The difference was ≤15%, indicating that the assay was within acceptable parameters. be.

[0132] Quantitative Analysis of Total ApoE:CSF Apolipoprotein E by LC-MS / MS Assay (ApoE) measures total ApoE levels in CSF. , trypsin digestion of the protein was performed, and the three isoforms (ApoE2, ApoE3, ApoE4, ApoE5, ApoE6, ApoE7, ApoE8, ApoE9, ApoE10, ApoE11, ApoE12, ApoE13, ApoE14, ApoE15, ApoE16, ApoE17, ApoE18, ApoE19, ApoE20, In all cases (ApoE3, ApoE4, and ApoE5), the unique peptides were expressed as a fraction of the total ApoE protein. CSF ApoE samples were used as a surrogate for measuring protein concentrations. Thermo connected to a Fisher Quantiva triple quadrupole mass spectrometer Tandem mass spectrometry using the Aria Cohesive TLX-4 high-flow LC The data are monitored by multiple reaction monitoring (MRM). and Thermo Fisher LC Quan data analysis software. The following ions were measured:

[0133] [Table 12]

[0134] The limit of detection (LOD) for CSF ApoE was 0.33 μg / mL. The limit of CSF ApoE is determined as 1.0 μg / mL. mL.

[0135] All publications, patents, and patent applications, and other documents described or cited herein and the content of electronically available information, each publication is incorporated by reference. No. 6,027,797, filed Dec. 1, 2004, and entitled "Patent Document No. 10-110049," which is incorporated herein by reference in its entirety to the same extent as if specifically and individually indicated. Applicants reserve the right to modify, translate, translate and distribute any such documents, patents, patent applications, or other physical and electronic materials. The right to physically incorporate into this application any and all materials and information from the document is reserved. do.

[0136] The methods illustratively described herein may be used in combination with any one element not specifically disclosed herein. Alternatively, the practice may be suitably carried out in the absence of one or more elements, one or more limitations. For example, the terms "comprising," "including," and "containing" "Ining" and the like should be interpreted expansively and without limitation. The terms and expressions used are intended to be words of description and not of limitation. and in the use of such terms and expressions, any equivalent of the properties shown and described. There is no intention to exclude any of the foregoing or any portion thereof. Various modifications are intended to be within the scope of the claimed invention. It is recognized that the present invention is therefore directed to preferred embodiments and optional features. Modifications of the invention specifically disclosed by but incorporated into the invention disclosed herein. It is understood that modifications and variations may be made by those skilled in the art, and such modifications and variations are It should be understood that all such modifications are considered to be within the scope of the present invention.

[0137] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings included also form part of the method. The general description of the method with a condition or negative qualification that removes any of the subjects from the genus is The excised material is included whether or not specifically listed herein. do.

[0138] Other embodiments are within the scope of the following claims. is described by a Markush group, one skilled in the art would recognize that the invention is thereby Neither individual members nor subgroups of members of the Kashmiri group are described. Recognize that there is.

Claims

1. 1. A method for determining an apolipoprotein E (ApoE) phenotype in a sample, comprising: (a) purifying ApoE in a sample; (b) ionizing the ApoE in the sample to obtain one or more ions of ApoE; generating (c) detecting ions from step (b) by mass spectrometry. and and wherein the ApoE alleles present in the sample are detected in step (c). The method is determined from the identity of the ions detected.

2. 10. The method of claim 1, wherein the purifying comprises liquid chromatography.

3. The liquid chromatography comprises high performance liquid chromatography (HPLC). The method of claim 2.

4. 10. The method of claim 1, wherein the purifying comprises solid phase extraction (SPE).

5. The method of claim 1 , wherein the ionization comprises electrospray ionization (ESI). Law.

6. 10. The method of claim 1, wherein the ionizing comprises ionizing in a positive mode. 。

7. The method of claim 1 further comprising adding an internal standard.

8. The method of claim 7 , wherein the internal standard is isotopically labeled.

9. The method of claim 1 , wherein the sample is cerebrospinal fluid (CSF).

10. The method of claim 1 , wherein the sample is serum.

11. 10. The method of claim 1, further comprising digesting the ApoE prior to purification.

12. The method of claim 11 , wherein the digestion comprises trypsin digestion.

13. 12. The method of claim 11, wherein the digestion comprises microwave digestion.

14. The method of claim 1, wherein the phenotype is ApoE2 / ApoE2.

15. The ApoE2 / ApoE2 phenotype was 665.72±0.5, 835.93±0.5 , 866.99±0.5, and 982.08±0.5 15. The method of claim 14, wherein the charge ratio is determined by the presence of fragment ions having the same charge ratio.

16. The method of claim 1, wherein the phenotype is ApoE2 / ApoE3.

17. The ApoE2 / ApoE3 phenotype was 374.42±0.5, 502.55±0.5 , 665.72±0.5, 835.93±0.5, 866.99±0.5, and 982. The presence of fragment ions having a mass / charge ratio selected from the group consisting of: 0.8±0.

5. The method of claim 16, wherein the presence of

18. The method of claim 1, wherein the phenotype is ApoE2 / ApoE4.

19. The ApoE2 / ApoE4 phenotype was 374.42±0.5, 502.55±0.5 、６４９．７４±０．５、６６５．７２±０．５、８３５．９３±０．５、８６６．９９ ±0.5, 892.96±0.5, and 982.08±0.5 19. The method of claim 18, wherein the fragment ions are determined by the presence of fragment ions having a mass / charge ratio of How to do it.

20. The method of claim 1, wherein the phenotype is ApoE3 / ApoE3.

21. The ApoE3 / ApoE3 phenotype was 374.42±0.5, 502.55±0.5 , 866.99±0.5, and 982.08±0.5 21. The method of claim 20, wherein the charge ratio is determined by the presence of fragment ions having a charge ratio of 0.01 to 0.

01.

22. The method of claim 1, wherein the phenotype is ApoE3 / ApoE4.

23. The ApoE3 / ApoE4 phenotype was 374.42±0.5, 502.55±0.5 , 649.74±0.5, 866.99±0.5, 892.96±0.5, and 982. The presence of fragment ions having a mass / charge ratio selected from the group consisting of: 0.8±0.

5.

23. The method of claim 22, wherein the presence of

24. The method of claim 1, wherein the phenotype is ApoE4 / ApoE4.

25. ApoE3 / ApoE4 phenotypes were 374.42±0.5, 502.55±0.5, and 6 49.74±0.5, and 892.96±0.5 mass / charge 25. The method of claim 24, wherein the ratio is determined by the presence of fragment ions having a ratio.

26. Claims that the presence of the ApoE4 allele indicates an increased risk of developing Alzheimer's disease Item 1. The method according to item 1.

27. The presence of the ApoE4 / ApoE4 allele indicates an increased risk of developing Alzheimer's disease. The method of claim 1 .

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