Detection of amyloid β by mass spectrometry

Abstract

Methods for detecting or quantifying amyloid beta are provided. In a specific aspect, methods for detecting amyloid beta or fragments thereof by mass spectrometry are provided herein. In another aspect, methods for determining the ratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40) are provided herein. In another aspect, methods for diagnosing or prognosing Alzheimer's disease or dementia are provided herein.

Description

[0001] This application is a divisional application. The application date of the original application is September 28, 2016, the application number is 2016800683335 (PCT / US2016 / 054148), and the name of the invention is "Detection of amyloid protein Β by mass spectrometry".

[0002] Cross-references to related patent applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 234,027, filed September 28, 2015, and U.S. Provisional Application No. 62 / 277,772, filed January 12, 2016, each of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to the detection or quantification of amyloid beta. In a particular aspect, the present invention relates to a method for the detection of amyloid beta or fragments thereof by mass spectrometry. Background Art

[0005] Alzheimer's disease is the most common form of dementia affecting the elderly population. Alzheimer's disease is characterized by a progressive decline in cognitive abilities, specifically, memory and learning. One of the hallmarks of the disease is neuritic plaques composed of amyloid beta (Aβ or Abeta) peptides.

[0006] Current clinical diagnostic methods for predicting or diagnosing Alzheimer's disease have low accuracy and sensitivity. Immunoassays are currently available to detect amyloid beta, which is a biomarker that predicts progression to Alzheimer's disease. However, there is concern about the inter-laboratory variability in the results observed using currently available immunoassays.

[0007] There is a need for accurate and sensitive assays for detecting amyloid beta. Summary of the invention

[0008] Provided herein are methods for detecting or determining the amount of amyloid beta (Aβ) in a sample by mass spectrometry, including tandem mass spectrometry.

[0009] In certain embodiments, the method provided herein for determining the amount of amyloid β comprises (a) purifying amyloid β in a sample; (b) ionizing amyloid β in the sample; and (c) determining the amount of amyloid β ion(s) by mass spectrometry; wherein the amount of amyloid β ion(s) is correlated with the amount of amyloid β in the sample.

[0010] In certain embodiments, the method provided herein for determining the amount of amyloid β comprises (a) purifying amyloid β in a sample; (b) ionizing amyloid β in the sample to produce precursor ions of amyloid β; (c) generating one or more fragment ions of amyloid β; and (d) determining the amount of ion (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of amyloid β ion (one or more) is related to the amount of amyloid β in the sample.

[0011] In certain embodiments, the method provided herein for determining the amount of amyloid β comprises (a) digesting amyloid β in a sample to generate one or more fragments of amyloid β; (b) purifying one or more amyloid β fragments; (c) ionizing one or more amyloid β fragments to produce precursor ions; (d) generating one or more fragment ions; and (e) determining the amount of ion (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of the ion (one or more) is related to the amount of the amyloid β fragment (one or more) in the sample.

[0012] In certain embodiments, provided herein is a method for determining the amount of amyloid β42 (Aβ42). In some embodiments, the Aβ42 fragment comprises the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4). In some embodiments, the method comprises (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42); (b) purifying Aβ42; (c) ionizing Aβ42 to generate precursor ions; (d) generating one or more fragment ions of Aβ42; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ42 in the sample.

[0013] In certain embodiments, provided herein is a method for determining the amount of amyloid beta 40 (Aβ40). In some embodiments, the Aβ40 fragment comprises the sequence GAIIGLMVGGVV (SEQ ID NO: 2). In some embodiments, the method comprises (a) digesting amyloid beta in a sample to generate amyloid beta 40 (Aβ40); (b) purifying Aβ40; (c) ionizing Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ40 in the sample.

[0014] In certain embodiments, provided herein are methods for determining the amount of amyloid β42 (Aβ42) and amyloid β40 (Aβ40). In some embodiments, the method comprises (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42) and amyloid β40 (Aβ40); (b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ42 and Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ42 and Aβ40 in the sample.

[0015] In certain embodiments, provided herein is a method for determining the ratio of amyloid β42 (Aβ42) to amyloid β40 (Aβ40). In some embodiments, the method includes (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42) and amyloid β40 (Aβ40); (b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ42 and Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; and (f) determining the ratio of Aβ42 to Aβ40. In some embodiments, the method includes determining the ratio of Aβ40 to Aβ42.

[0016] In certain embodiments, provided herein is a method for diagnosing or prognosing Alzheimer's disease or dementia, the method comprising determining the amount of amyloid β in a test sample by mass spectrometry; wherein abnormal levels of amyloid β predict or diagnose Alzheimer's disease. In some embodiments, the method may include: (a) purifying amyloid β in a sample; (b) ionizing amyloid β in a sample; and (c) determining the amount of amyloid β ions (one or more) by mass spectrometry; and (d) the amount of amyloid β ions (one or more) is related to the amount of amyloid β in the sample; wherein abnormal levels of amyloid β predict or diagnose Alzheimer's disease. In some embodiments, the method comprises determining the ratio of amyloid β fragments. In some embodiments, the method comprises determining the ratio of amyloid β42 (Aβ42) to amyloid β40 (Aβ40). In some embodiments, the method comprises determining the ratio of amyloid β42 (Aβ40) to amyloid β40 (Aβ42).

[0017] In certain embodiments, the methods provided herein include pretreating the surface of the equipment in contact with the sample. In some embodiments, the pretreatment includes pre-coating the surface of the equipment with a reagent that prevents amyloid beta or its fragments from adhering to the surface. In some embodiments, the pretreatment includes pretreatment of bacterial lysate. In some embodiments, the pretreatment includes pretreatment of E. coli lysate. In some embodiments, the E. coli lysate includes trypsin-digested E. coli lysate. In some embodiments, the pretreated equipment includes but is not limited to test tubes or plates, pipette tips, sample preparation equipment, liquid chromatography equipment, and mass spectrometry equipment.

[0018] In certain embodiments, the methods provided herein include treating or incubating a sample with an agent that stabilizes amyloid β or a fragment thereof. In some embodiments, the methods provided herein include treating or incubating a sample with an amyloid β antibody. In some embodiments, the methods provided herein include treating or incubating a sample with at least two different amyloid β antibodies. In some embodiments, the amyloid β antibody includes an antibody that binds to the C-terminus of amyloid β. In some embodiments, the amyloid β antibody includes an antibody that binds to the N-terminus of amyloid β. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein E2. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein E4. In some embodiments, an agent that stabilizes amyloid β includes an antibody that binds to the C-terminus of amyloid β, an antibody that binds to the N-terminus of amyloid β, apolipoprotein E2, apolipoprotein E4, or a combination thereof. In some embodiments, an agent that stabilizes amyloid β provided herein provides stability of at least 1 month at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability for at least 2 months at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability for at least 3 months at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least two freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least three freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least four freeze-thaw cycles.

[0019] In certain embodiments, the methods provided herein include digesting amyloid β in a sample. In some embodiments, the methods provided herein include digesting amyloid β using an enzyme. In some embodiments, the enzyme is Lys-C. In some embodiments, the methods provided herein include digesting amyloid β using urea. In some embodiments, urea is at a concentration suitable for protein digestion. In some embodiments, urea is 6M urea. In some embodiments, the methods provided herein include digesting amyloid β using urea and Lys-C. In some embodiments, digestion includes digesting under conditions that reduce digestion time or increase digestion efficiency. In some embodiments, digestion includes digesting in a microwave. In some embodiments, the methods provided herein include measuring the amount of digested amyloid β.

[0020] In certain embodiments, the methods provided herein include extraction. In some embodiments, the methods provided herein include mixed mode anion exchange extraction. In some embodiments, the methods provided herein include solid phase extraction.

[0021] In certain embodiments, the methods provided herein comprise eluting and drying the sample using heated nitrogen.In some embodiments, the sample is resuspended in a reconstitution buffer.

[0022] In certain embodiments, the purified sample comprises liquid chromatography. In some embodiments, liquid chromatography includes but is not limited to reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC). In a preferred embodiment, liquid chromatography comprises HPLC. In some embodiments, the HPLC column generally includes medium (that is, packing material) to promote the separation (that is, classification) of chemical part. Suitable column can include C-4, C-8, C-12 or C-18 column. In a preferred embodiment, suitable HPLC column is C-4 column.

[0023] In certain embodiments, the methods provided herein include using a device that reduces the adhesion of amyloid beta to the surface of the device. In some embodiments, the device includes PEEK (polyetheretherketone) tubing or equipment. In some embodiments, the device includes metal tubing or equipment.

[0024] In certain embodiments, the methods provided herein include tandem mass spectrometry. In some embodiments, the methods provided herein include ionizing in positive mode. In some embodiments, the methods provided herein include ionizing in negative mode. In some embodiments, the methods provided herein include ionizing using heated electrospray ionization (HESI). In some embodiments, the methods provided herein include ionizing using electrospray ionization (ESI). In some embodiments, the methods provided herein include ionizing using atmospheric pressure chemical ionization (APCI). In a preferred embodiment, the methods provided herein include ionizing using heated electrospray ionization (HESI) in positive mode. In some embodiments, the collision energy is between 5V and 60V. In some embodiments, the collision energy is between 10V and 50V. In some embodiments, the collision energy is between 20V and 50V. In some embodiments, the collision energy is between 20V and 45V.

[0025] In certain embodiments, the methods provided herein include detecting or measuring the amount of amyloid beta 40 (Aβ40). In some embodiments, Aβ40 includes the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 1). In some embodiments, the methods provided herein include detecting or measuring the amount of a fragment of Aβ40. In some embodiments, the Aβ40 fragment includes the sequence GAIIGLMVGGVV (SEQ ID NO: 2). In some embodiments, the Aβ40 fragment includes a sequence comprising an N-terminal or C-terminal winged peptide. In some embodiments, the Aβ40 fragment includes SEQID NO: 2 and an N-terminal or C-terminal winged peptide. In some embodiments, the winged peptide is hydrophilic. In some embodiments, the winged peptide includes at least one amino acid. In some embodiments, the winged peptide includes at least two amino acids. In some embodiments, the winged peptide includes at least three amino acids. In some embodiments, the winged peptide includes at least four amino acids. In some embodiments, the winged peptide includes at least five amino acids. In some embodiments, the winged peptide comprises at least six amino acids. In some embodiments, the amount of the Aβ40 fragment correlates to the amount of Aβ40 in the sample.

[0026] In certain embodiments, the methods provided herein include detecting or measuring the amount of amyloid beta 42 (Aβ42). In some embodiments, Aβ42 includes the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQID NO: 3). In some embodiments, the methods provided herein include detecting or measuring the amount of a fragment of Aβ42. In some embodiments, the Aβ42 fragment includes the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4). In some embodiments, the Aβ42 fragment includes a sequence comprising an N-terminal or C-terminal wing peptide. In some embodiments, the Aβ42 fragment includes SEQ ID NO: 4 and an N-terminal or C-terminal wing peptide. In some embodiments, the wing peptide is hydrophilic. In some embodiments, the wing peptide includes at least one amino acid. In some embodiments, the wing peptide includes at least two amino acids. In some embodiments, the wing peptide includes at least three amino acids. In some embodiments, the wing peptide includes at least four amino acids. In some embodiments, the wing peptide includes at least five amino acids. In some embodiments, the wing peptide comprises at least six amino acids. In some embodiments, the amount of the Aβ42 fragment correlates to the amount of Aβ42 in the sample.

[0027] In certain embodiments, the methods provided herein include detecting or determining the ratio of Aβ40 to Aβ42 (Aβ40:Aβ42). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ40 fragments to Aβ42 fragments (Aβ40 fragments:Aβ42 fragments). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ42 to Aβ40 (Aβ42:Aβ40). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ42 fragments to Aβ40 fragments (Aβ42 fragments:Aβ40 fragments).

[0028] In certain embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.6 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.5 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.45 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.4 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.35 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.3 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.25 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.2 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.15 or less predicts or diagnoses Alzheimer's disease.

[0029] In certain embodiments, the method comprises 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 comprise generating one or more fragment ions of Aβ or a fragment thereof. In some embodiments, at least one of the fragment ions has a mass / charge ratio of 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.

[0030] In certain embodiments, the methods provided herein include the addition of an internal standard. In some embodiments, the internal standard includes an isotopically labeled internal standard. In some embodiments, the internal standard includes 13 C 15 In some embodiments, the internal standard includes at least one 13 C 15In some embodiments, at least one of the precursor ions of the internal standard has a mass / charge ratio of 1110.7±0.5. In some embodiments, the method can include generating one or more fragment ions of the internal standard. In some embodiments, at least one of the fragment ions has a mass / charge ratio of 768.48±0.5, 825.5±0.5, or 882.52±0.5.

[0031] 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 some embodiments, the limit of quantitation of the method is less than or equal to 1 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.5 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.2 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.1 ng / mL.

[0032] 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 or equal to 0.05 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.01 ng / mL.

[0033] In some embodiments, amyloid beta is not derivatized prior to mass spectrometry. In some embodiments, amyloid beta is derivatized prior to mass spectrometry.

[0034] 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 sample is saliva or urine.

[0035] In some embodiments, a method can include adding a reagent to a sample in an amount sufficient to deproteinize the sample.

[0036] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a protein" includes a plurality of protein molecules.

[0037] As used herein, the term "purification" or "purifying" does not refer to the removal of all materials except the analyte(s) of interest from a sample. Rather, purification refers to the process of enriching the amount of one or more analytes of interest relative to other components in the sample that may interfere with the detection of the analyte(s) of interest. Samples are purified herein by a variety of means to allow for the removal of one or more interfering substances, e.g., one or more substances that would interfere with the detection of selected amyloid β parent and daughter ions by mass spectrometry.

[0038] As used herein, the term "test sample" refers to any sample that may contain amyloid beta. As used herein, the term "body fluid" means any fluid that can be isolated from an individual's body. For example, "body fluid" may include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc.

[0039] As used herein, the term "derivatization" means reacting two molecules to form a new molecule. The derivatizing agent may include an isothiocyanate group, a dinitro-fluorophenyl group, a nitrophenoxycarbonyl group, and / or a phthalaldehyde group, and the like.

[0040] As used herein, the term "chromatography" refers to the process by which a liquid or gas-borne chemical mixture separates into its components due to the differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0041] As used herein, the term "liquid chromatography" or "LC" means the process of selectively retarding one or more components of a fluid solution as the fluid uniformly permeates through a column of finely divided material or through a capillary channel. The retarding is caused by the distribution of the components of the mixture between the one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), and high turbulence liquid chromatography (HTLC).

[0042] As used herein, the term "high performance liquid chromatography" or "HPLC" refers to a liquid chromatography method that increases the degree of separation by forcing a mobile phase under pressure through a stationary phase, usually a densely packed column.

[0043] As used herein, the term "high turbulence liquid chromatography" or "HTLC" refers to a form of chromatography that utilizes turbulence of the material being tested through a column packing as the basis for separation. HTLC has been applied to prepare samples containing two unknown drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further describe HTLC. "Turbulent flow" is understood by one of ordinary skill in the art. When a fluid flows slowly and smoothly, the flow is referred to as "laminar flow." For example, a fluid moving through an HPLC column at a low flow rate is laminar. In laminar flow, the motion of fluid particles is orderly, with the particles moving generally in a straight line. At faster speeds, the inertia of the water overcomes the fluid friction and produces turbulence. A fluid that is not in contact with an irregular boundary "outruns" a fluid that is slowed by friction or deflected by a rough surface. When a fluid flows turbulently, it flows in eddies and rotations (or vortices) that have a greater "drag force" than when the flow is laminar. Many references are available to help determine when a fluid flow is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction ,PSBernard&J.M.Wallace,John Wiley&Sons,Inc.,(2000); An Introduction to Turbulent Flow ,Jean Mathieu&Julian Scott,Cambridge University Press(2001)).

[0044] As used herein, the term "gas chromatography" or "GC" refers to a chromatography method in which a sample mixture is vaporized and injected into a stream of a carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or particulate solid, and separated into its component compounds according to their affinity for the stationary phase.

[0045] As used herein, the term "large particle column" or "extraction column" refers to a chromatographic column comprising an average particle size greater than about 35 μm. As used in this context, the term "about" means ± 10%. In a preferred embodiment, the column comprises particles having a diameter of about 60 μm.

[0046] As used herein, the term "analytical column" refers to a chromatographic column having enough chromatographic plates to achieve separation of material in a sample eluted from the column, said separation being sufficient to allow determination of the presence or amount of an analyte. Such a column is generally distinguished from an "extraction column" which has the general purpose of separating or extracting retained material from non-retained material in order to obtain a purified sample for further analysis. As used in this context, the term "approximately" means ±10%. In a preferred embodiment, the analytical column comprises particles having a diameter of approximately 4 μm.

[0047] As used herein, the terms "on-line" and "inline", e.g., as used in "online automated mode" or "online extraction", refer to procedures that are performed without the need for operator intervention. In contrast, the term "off-line" as used herein refers to procedures that require manual intervention by an operator. Thus, if a sample undergoes precipitation, and the supernatant is then manually loaded into an autosampler, the precipitation and loading steps are off-line from subsequent steps. In various embodiments of the method, one or more steps may be performed in an online automated manner.

[0048] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies a compound by its mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. The compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometer, where, due to a combination of magnetic and electric fields, the ions follow a path in space that depends on mass ("m") and charge ("z"). See, e.g., U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces”; U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem Mass Spectrometry”; U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry”; U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes”; Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000).

[0049] As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods that generate and detect negative ions. As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods that generate and detect positive ions.

[0050] As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions having a net charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

[0051] As used herein, the term "electron ionization" or "EI" refers to a process by which an analyte of interest in the gas or vapor phase interacts with a flow of electrons. The collision of the electrons with the analyte produces analyte ions that can then be subjected to mass spectrometry techniques.

[0052] As used herein, the term "chemical ionization" or "CI" refers to a process in which a reagent gas (eg, ammonia) is subjected to electron impacts, and analyte ions are formed by the interaction of the reagent gas ions and analyte molecules.

[0053] As used herein, the term "fast atom bombardment" or "FAB" refers to a method in which a beam of high energy atoms (usually, Xe or Ar) impacts a non-volatile sample, desorbing and ionizing the molecules contained in the sample. The test sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine. The selection of an appropriate matrix for a compound or sample is an empirical process.

[0054] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to a method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through a variety of ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy absorbing matrix, which promotes the desorption of analyte molecules.

[0055] As used herein, the term "surface enhanced laser desorption ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through a variety of ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process can also employ energy absorbing materials to facilitate ionization.

[0056] As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution is passed along a short capillary length to which a high positive or negative potential is applied at the end. The solution arriving at the end of the tube is vaporized (atomized) into a jet or spray of very small solution droplets in a solvent vapor. This droplet flows through an evaporation chamber, which is slightly heated to prevent condensation and to evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until the time when the natural repulsion between like charges causes the release of ions and neutral molecules.

[0057] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI; however, APCI produces ions by ion-molecule reactions occurring within a plasma at atmospheric pressure. The plasma is maintained by a discharge between a spray capillary and a counter electrode. The ions are then extracted into a mass analyzer, typically by using a set of differentially pumped skimmer stages. A countercurrent of dry and preheated N2 gas can be used to improve solvent removal. Gas phase ionization in APCI can be used more effectively than ESI for the analysis of low polarity species.

[0058] As used herein, the term "atmospheric pressure photoionization" or "APPI" refers to a form of mass spectrometry in which the mechanism of photoionization of a molecule M is photon absorption and electron emission to form a molecular ion M+. Because the photon energy is usually just above the ionization potential, the molecular ion is not prone to dissociation. In many cases, it is possible to analyze the sample without the need for chromatography, thereby saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur when M has a high proton affinity. Because the sum of M+ and MH+ is a constant, this does not affect quantitative accuracy. Drug compounds in protic solvents are usually observed as MH+, while non-polar compounds such as naphthalene or testosterone usually form M+. Robb, DB, Covey, TR and Bruins, AP (2000): See, e.g., Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography-mass spectrometry. Anal. Chem. 72(15): 3653-3659.

[0059] As used herein, the term "inductively coupled plasma" or "ICP" refers to a process in which a sample interacts with a partially ionized gas at a sufficiently high temperature so that most of the elements are atomized and ionized.

[0060] As used herein, the term "field desorption" refers to a method in which a non-volatile test sample is placed on an ionizing surface and a strong electric field is used to generate analyte ions.

[0061] As used herein, the term "desorption" refers to the removal of an analyte from a surface and / or the passage of an analyte into the gas phase.

[0062] As used herein, the term "limit of quantification," "limit of quantitation," or "LOQ" refers to the point at which a measurement becomes quantitatively meaningful. The analyte response at this LOQ is discernible, discrete, and reproducible with a precision of 20% and an accuracy of 80% to 120%.

[0063] As used herein, the term "limit of detection" or "LOD" is the point at which a measurement is greater than the uncertainty associated with it. The LOD is arbitrarily defined as 2 standard deviations (SD) from zero concentration.

[0064] As used herein, the "amount" of amyloid β in a body fluid sample generally refers to an absolute value reflecting the mass of amyloid β detectable in a volume of body fluid. However, the amount also contemplates a relative amount compared to another amount of amyloid β. For example, the amount of amyloid β in a body fluid can be an amount that is greater or less than a control or normal level of amyloid β normally present.

[0065] As used herein with respect to quantitative measurements - measurements that do not include the mass of an ion - the term "about" refers to the indicated value plus or minus 10%. Mass spectrometers can vary slightly in determining the mass of a given analyte. In the context of the mass of an ion or the mass / charge ratio of an ion, the term "about" refers to + / - 0.50 atomic mass units.

[0066] The above description of the invention is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The sequences of Aβ40 (SEQ ID NO: 1), Aβ40 fragment (SEQ ID NO: 2), Aβ42 (SEQ ID NO: 3), and Aβ42 fragment (SEQ ID NO: 4) are shown.

[0068] Figure 2 Liquid chromatography method information is shown. Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in ACN.

[0069] Figure 3 Heated electrospray ionization (HESI) source conditions are shown.

[0070] Figure 4 The linear range of the Aβ42 analysis is shown.

[0071] Figure 5 The linear range of the Aβ40 analysis is shown.

[0072] Figure 6 and7 Example patient chromatograms are shown for Aβ40, Aβ42, and internal standard.

[0073] Figure 8 The ratio of Aβ42:Aβ40 in 211 subjects, including 91 Alzheimer's disease patients, 66 borderline Alzheimer's disease patients, and normal subjects, is shown.

[0074] Fig. 9 Shown is the ratio of Aβ42:Aβ40 in female versus male Alzheimer's disease patients.

[0075] Fig.10 Shown is the ratio of Aβ42:Aβ40 in age-stratified groups of Alzheimer's disease patients.

[0076] Fig.11 Shown is a sensitivity comparison between C8 vs. C4 analytical columns.

[0077] Fig.12 Recovery of Aβ40 from treated versus untreated tubes is shown.

[0078] Fig.13 Recovery of Aβ42 from treated versus untreated tubes is shown.

[0079] Fig.14 Shown are Aβ values ​​(pg / mL) for patients categorized by decreasing Aβ40 levels.

[0080] Fig.15 Shown are Aβ values ​​(pg / mL) for patients categorized by decreasing Aβ42 levels.

[0081] Fig.16 Patients' Alzheimer's disease diagnosis categorized by Aβ42 / Aβ40 values ​​is shown.

[0082] Fig.17 Patient Alzheimer's disease diagnosis based on the Aβ42 / Aβ40 ratio is shown. DETAILED DESCRIPTION

[0083] Provided herein are methods for detecting or determining the amount of amyloid β (Aβ) in a sample by mass spectrometry, including tandem mass spectrometry. In certain embodiments, provided herein are methods for determining the amount of amyloid β, comprising (a) purifying the amyloid β in the sample; (b) ionizing the amyloid β in the sample; and (c) determining the amount of the amyloid β ion (one or more) by mass spectrometry; wherein the amount of the amyloid β ion (one or more) is related to the amount of the amyloid β in the sample.

[0084] In certain embodiments, the method provided herein for determining the amount of amyloid β comprises (a) purifying amyloid β in a sample; (b) ionizing amyloid β in the sample to produce precursor ions of amyloid β; (c) generating one or more fragment ions of amyloid β; and (d) determining the amount of ion (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of amyloid β ion (one or more) is related to the amount of amyloid β in the sample.

[0085] In certain embodiments, the method provided herein for determining the amount of amyloid β comprises (a) digesting amyloid β in a sample to generate one or more fragments of amyloid β; (b) purifying one or more amyloid β fragments; (c) ionizing amyloid β in the sample to produce precursor ions of amyloid β; (d) generating one or more fragment ions of amyloid β; and (e) determining the amount of ion (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of amyloid β ion (one or more) is related to the amount of amyloid β in the sample.

[0086] In certain embodiments, provided herein is a method for determining the amount of amyloid β42 (Aβ42). In some embodiments, the Aβ42 fragment comprises the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4). In some embodiments, the method comprises (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42); (b) purifying Aβ42; (c) ionizing Aβ42 to generate precursor ions; (d) generating one or more fragment ions of Aβ42; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ42 in the sample.

[0087] In certain embodiments, provided herein is a method for determining the amount of amyloid beta 40 (Aβ40). In some embodiments, the Aβ40 fragment comprises the sequence GAIIGLMVGGVV (SEQ ID NO: 2). In some embodiments, the method comprises (a) digesting amyloid beta in a sample to generate amyloid beta 40 (Aβ40); (b) purifying Aβ40; (c) ionizing Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ40 in the sample.

[0088] In certain embodiments, provided herein are methods for determining the amount of amyloid β42 (Aβ42) and amyloid β40 (Aβ40). In some embodiments, the method comprises (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42) and amyloid β40 (Aβ40); (b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ42 and Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; wherein the amount of ions (one or more) is related to the amount of Aβ42 and Aβ40 in the sample.

[0089] In certain embodiments, provided herein is a method for determining the ratio of amyloid β42 (Aβ42) to amyloid β40 (Aβ40). In some embodiments, the method includes (a) digesting amyloid β in a sample to generate amyloid β42 (Aβ42) and amyloid β40 (Aβ40); (b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to generate precursor ions; (d) generating one or more fragment ions of Aβ42 and Aβ40; and (e) determining the amount of ions (one or more) from step (c) or (d) or both by mass spectrometry; and (f) determining the ratio of Aβ42 to Aβ40. In some embodiments, the method includes determining the ratio of Aβ40 to Aβ42.

[0090] In certain embodiments, provided herein are methods for diagnosing or prognosing Alzheimer's disease or dementia, the methods comprising determining the amount of amyloid β in a test sample by mass spectrometry; wherein abnormal levels of amyloid β predict or diagnose Alzheimer's disease. In some embodiments, the methods may include: (a) purifying amyloid β in a sample; (b) ionizing amyloid β in a sample; and (c) determining the amount of amyloid β ion (one or more) by mass spectrometry; and (d) the amount of amyloid β ion (one or more) is related to the amount of amyloid β in the sample; wherein abnormal levels of amyloid β predict or diagnose Alzheimer's disease. In some embodiments, the methods include determining the ratio of amyloid β fragments. In some embodiments, the methods include determining the ratio of amyloid β42 (Aβ42) to amyloid β40 (Aβ40). In some embodiments, the methods include determining the ratio of amyloid β42 (Aβ40) to amyloid β40 (Aβ42).

[0091] In certain embodiments, the methods provided herein include pretreating the surface of the equipment in contact with the sample. In some embodiments, the pretreatment includes pre-coating the surface of the equipment with an agent that prevents amyloid beta or its fragments from adhering to the surface. In some embodiments, the pretreatment includes bacterial lysate pretreatment. In some embodiments, the pretreatment includes E. coli lysate pretreatment. In some embodiments, the E. coli lysate includes trypsin-digested E. coli lysate. In some embodiments, the pretreated equipment includes but is not limited to test tubes or plates, pipette tips, sample preparation equipment, liquid chromatography equipment, and mass spectrometry equipment.

[0092] In certain embodiments, the methods provided herein include treating or incubating a sample with an agent that stabilizes amyloid β or a fragment thereof. In some embodiments, the methods provided herein include treating or incubating a sample with an amyloid β antibody. In some embodiments, the methods provided herein include treating or incubating a sample with at least two different amyloid β antibodies. In some embodiments, the amyloid β antibody includes an antibody that binds to the C-terminus of amyloid β. In some embodiments, the amyloid β antibody includes an antibody that binds to the N-terminus of amyloid β. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein E2. In some embodiments, an agent that stabilizes amyloid β includes apolipoprotein E4. In some embodiments, an agent that stabilizes amyloid β includes an antibody that binds to the C-terminus of amyloid β, an antibody that binds to the N-terminus of amyloid β, apolipoprotein E2, apolipoprotein E4, or a combination thereof. In some embodiments, an agent that stabilizes amyloid β provided herein provides stability of at least 1 month at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability for at least 2 months at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability for at least 3 months at -70°C. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least two freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least three freeze-thaw cycles. In some embodiments, the reagents for stabilizing amyloid β provided herein give stability through at least four freeze-thaw cycles.

[0093] In certain embodiments, the methods provided herein include digesting amyloid β in a sample. In some embodiments, the methods provided herein include digesting amyloid β using an enzyme. In some embodiments, the enzyme is Lys-C. In some embodiments, the methods provided herein include digesting amyloid β using urea. In some embodiments, urea is at a concentration suitable for protein digestion. In some embodiments, urea is 6M urea. In some embodiments, the methods provided herein include digesting amyloid β using urea and Lys-C. In some embodiments, digestion includes digesting under conditions that reduce digestion time or increase digestion efficiency. In some embodiments, digestion includes digesting in a microwave.

[0094] In certain embodiments, the methods provided herein include extraction. In some embodiments, the methods provided herein include mixed mode anion exchange extraction. In some embodiments, the methods provided herein include solid phase extraction.

[0095] In certain embodiments, the methods provided herein comprise eluting and drying the sample using heated nitrogen.In some embodiments, the sample is resuspended in a reconstitution buffer.

[0096] In certain embodiments, the purified sample comprises liquid chromatography. In some embodiments, liquid chromatography includes but is not limited to reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC). In a preferred embodiment, liquid chromatography comprises HPLC. In some embodiments, the HPLC column generally includes medium (that is, packing material) to promote the separation (that is, classification) of chemical part. Suitable column can include C-4, C-8, C-12 or C-18 column. In a preferred embodiment, suitable HPLC column is C-4 column.

[0097] In certain embodiments, the methods provided herein include using a device that reduces the adhesion of amyloid beta to the surface of the device. In some embodiments, the device includes PEEK (polyetheretherketone) tubing or equipment. In some embodiments, the device includes metal tubing or equipment.

[0098] In certain embodiments, the methods provided herein include tandem mass spectrometry. In some embodiments, the methods provided herein include ionizing in positive mode. In some embodiments, the methods provided herein include ionizing in negative mode. In some embodiments, the methods provided herein include ionizing using heated electrospray ionization (HESI). In some embodiments, the methods provided herein include ionizing using electrospray ionization (ESI). In some embodiments, the methods provided herein include ionizing using atmospheric pressure chemical ionization (APCI). In a preferred embodiment, the methods provided herein include ionizing using heated electrospray ionization (HESI) in positive mode. In some embodiments, the collision energy is between 5V and 60V. In some embodiments, the collision energy is between 10V and 50V. In some embodiments, the collision energy is between 20V and 50V. In some embodiments, the collision energy is between 20V and 45V.

[0099] In certain embodiments, the methods provided herein include detecting or measuring the amount of amyloid beta 40 (Aβ40). In some embodiments, Aβ40 includes the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 1). In some embodiments, the methods provided herein include detecting or measuring the amount of a fragment of Aβ40. In some embodiments, the Aβ40 fragment includes the sequence GAIIGLMVGGVV (SEQ ID NO: 2). In some embodiments, the Aβ40 fragment includes a sequence comprising an N-terminal or C-terminal wing peptide. In some embodiments, the Aβ40 fragment includes SEQ ID NO: 2 and an N-terminal or C-terminal wing peptide. In some embodiments, the wing peptide is hydrophilic. In some embodiments, the wing peptide includes at least one amino acid. In some embodiments, the wing peptide includes at least two amino acids. In some embodiments, the wing peptide includes at least three amino acids. In some embodiments, the wing peptide includes at least four amino acids. In some embodiments, the wing peptide includes at least five amino acids. In some embodiments, the winged peptide comprises at least six amino acids. In some embodiments, the amount of the Aβ40 fragment correlates to the amount of Aβ40 in the sample.

[0100] In certain embodiments, the methods provided herein include detecting or measuring the amount of amyloid beta 42 (Aβ42). In some embodiments, Aβ42 includes the sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQID NO: 3). In some embodiments, the methods provided herein include detecting or measuring the amount of a fragment of Aβ42. In some embodiments, the Aβ42 fragment includes the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4). In some embodiments, the Aβ42 fragment includes a sequence comprising an N-terminal or C-terminal wing peptide. In some embodiments, the Aβ42 fragment includes SEQ ID NO: 4 and an N-terminal or C-terminal wing peptide. In some embodiments, the wing peptide is hydrophilic. In some embodiments, the wing peptide includes at least one amino acid. In some embodiments, the wing peptide includes at least two amino acids. In some embodiments, the wing peptide includes at least three amino acids. In some embodiments, the wing peptide includes at least four amino acids. In some embodiments, the wing peptide includes at least five amino acids. In some embodiments, the wing peptide comprises at least six amino acids. In some embodiments, the amount of the Aβ42 fragment correlates to the amount of Aβ42 in the sample.

[0101] In certain embodiments, the methods provided herein include detecting or determining the ratio of Aβ40 to Aβ42 (Aβ40:Aβ42). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ40 fragments to Aβ42 fragments (Aβ40 fragments:Aβ42 fragments). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ42 to Aβ40 (Aβ42:Aβ40). In some embodiments, the methods provided herein include detecting or determining the ratio of Aβ42 fragments to Aβ40 fragments (Aβ42 fragments:Aβ40 fragments).

[0102] In certain embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.6 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.5 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.45 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.4 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.35 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.3 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.25 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.2 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.19 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.18 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.17 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.16 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.15 or less predicts or diagnoses Alzheimer's disease. In some embodiments, a ratio of Aβ42 to Aβ40 or a ratio of Aβ42 fragments to Aβ40 fragments of 0.1 or less predicts or diagnoses Alzheimer's disease.

[0103] In certain embodiments, the method comprises 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 comprise generating one or more fragment ions of Aβ or a fragment thereof. In some embodiments, at least one of the fragment ions has a mass / charge ratio of 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.

[0104] In certain embodiments, the methods provided herein include the addition of an internal standard. In some embodiments, the internal standard includes an isotopically labeled internal standard. In some embodiments, the internal standard includes 13 C 15 In some embodiments, the internal standard includes at least one 13 C 15 In some embodiments, at least one of the precursor ions of the internal standard has a mass / charge ratio of 1110.7±0.5. In some embodiments, the method can include generating one or more fragment ions of the internal standard. In some embodiments, at least one of the fragment ions has a mass / charge ratio of 768.48±0.5, 825.5±0.5, or 882.52±0.5.

[0105] 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 some embodiments, the limit of quantitation of the method is less than or equal to 1 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.5 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.2 ng / mL. In some embodiments, the limit of quantitation of the method is less than or equal to 0.1 ng / mL.

[0106] 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 or equal to 0.05 ng / mL. In some embodiments, the detection limit of the method is less than or equal to 0.01 ng / mL.

[0107] In some embodiments, amyloid beta is not derivatized prior to mass spectrometry. In some embodiments, amyloid beta is derivatized prior to mass spectrometry.

[0108] 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 sample is saliva or urine.

[0109] Suitable test samples include any test sample that may contain an analyte of interest. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source such as an animal, cell culture, or organ culture, etc. In certain preferred embodiments, the sample is obtained from a mammal such as a dog, a cat, a horse, etc. Particularly preferred mammals are primates, most preferably, men or women. Particularly preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or other tissue samples. For example, such a sample can be obtained from a patient; that is, a living person, male or female, who is himself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, serum.

[0110] Sample preparation for mass spectrometry

[0111] Methods that can be used to enrich amyloid β relative to other components (e.g., proteins) in a sample include, for example, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis (including capillary electrophoresis), affinity separation (including immunoaffinity separation), extraction methods (including ethyl acetate extraction and methanol extraction), and the use of chaotropic agents, or any combination of the above.

[0112] Protein precipitation is a preferred method for preparing a test sample. Such protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatography B 785:263-275 (2003) describes a protein precipitation technique suitable for this method. Protein precipitation can be used to remove most of the protein from the sample, leaving amyloid beta in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated protein. The resulting supernatant can then be applied to liquid chromatography and subsequent mass spectrometry. In certain embodiments, for example, protein precipitation such as acetonitrile protein precipitation is used before HPLC and mass spectrometry to eliminate the need for high turbulence liquid chromatography (HTLC) or other online extraction. Therefore, in such an embodiment, the method involves (1) performing protein precipitation of a sample of interest; and (2) loading the supernatant directly onto an HPLC-mass spectrometer without using online extraction or high turbulence liquid chromatography (HTLC).

[0113] In some preferred embodiments, HPLC, individually or in combination with one or more purification methods, can be used to purify amyloid beta before mass spectrometry. In such an embodiment, before ionization, the sample can be extracted using the HPLC extraction cartridge that captures the analyte, and then eluted and chromatographically on the second HPLC column or analytical HPLC column. Because the step related to these chromatographic procedures can be connected in an automated manner, the needs for operator participation during the purified analyte can be minimized. This feature can result in saving time and cost, and eliminates the possibility of operator error.

[0114] It is believed that turbulence, such as the turbulence provided by HTLC columns and methods, can enhance the rate of mass transfer and improve separation characteristics. HTLC columns separate components by means of high chromatographic flow rates through packed columns containing rigid particles. By adopting high flow rates (e.g., 3-5 mL / min), turbulence occurs in the column causing almost thorough interaction between the stationary phase and the analyte of interest (one or more). The advantage of using HTLC columns is that the macromolecular aggregation associated with the biological fluid matrix is ​​avoided due to not retaining high molecular weight species under turbulent conditions. The HTLC method combining multiple separations in one program reduces the need for lengthy sample preparation and operates at a significantly larger speed. Such a method also achieves separation performance that is superior to laminar flow (HPLC) chromatography. HTLC allows direct injection of biological samples (blood plasma, urine, etc.). Direct injection is difficult to achieve in traditional forms of chromatography, because denatured proteins and other biological debris rapidly block the separation column. HTLC also allows a very low sample volume of less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, preferentially 0.1 mL.

[0115] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry have been described elsewhere. See, e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. In certain embodiments of the method, the sample is subjected to protein precipitation as described above before being loaded on the HTLC column; in an optional preferred embodiment, the sample can be loaded directly on the HTLC without being subjected to protein precipitation. The HTLC extraction column is preferably a large particle column. In various embodiments, one of the various steps of the method can be performed in an online automated manner. For example, in one embodiment, steps (i)-(v) are performed in an online automated manner. In another, the steps of ionization and detection are performed online after steps (i)-(v).

[0116] Liquid chromatography (LC) including high performance liquid chromatography (HPLC) relies on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packings, wherein the laminar flow of the sample by the column is the basis for separating the analyte of interest from the sample. The technician will understand that the separation in such a column is a diffusion process. HPLC has been successfully applied to separate compounds in biological samples, but requires significant sample preparation before the separation and subsequent analysis using a mass spectrometer (MS), which makes this technology labor-intensive. In addition, most HPLC systems do not utilize mass spectrometers to the greatest extent, which makes only an HPLC system be connected to a single MS instrument, causing the tedious time required for carrying out a large number of experiments.

[0117] Various methods have been described for using HPLC for sample clean-up prior to mass spectrometry analysis. See, for example, Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000).

[0118] Technicians can select HPLC instruments and columns suitable for use with amyloid beta. Chromatographic columns generally include a medium (i.e., a filling material) that promotes the separation (i.e., classification) of chemical moieties. The medium may include tiny particles. The particles include a binding surface that interacts with a variety of chemical moieties to promote the separation of chemical moieties. A suitable binding surface is a hydrophobic binding surface, such as an alkyl binding surface. The alkyl binding surface may include an alkyl bound to C-4, C-8, C-12, or C-18, preferably a group bound to C-18. The chromatographic column includes an inlet for receiving a sample and an outlet for discharging an effluent including a fractionated sample. In one embodiment, a sample (or a pre-purified sample) is applied to the column at the inlet, eluted with a solvent or a solvent mixture, and discharged at the outlet. Different solvent modes may be selected for eluting an analyte (one or more) of interest. For example, liquid chromatography may be performed using a gradient mode, an amorphous mode, or a polymorphic (i.e., mixed) mode. During chromatography, the separation of materials is affected by variables, such as the selection of an eluent (also referred to as "mobile phase"), an elution mode, a gradient condition, a temperature, etc.

[0119] In certain embodiments, the analyte can be purified by applying the sample to the column under conditions where the analyte of interest is reversibly retained by the column packing material, without retaining one or more other materials. In these embodiments, a first mobile phase condition that retains the analyte of interest by the column can be employed, and once the unretained material is washed through, a second mobile phase condition can be subsequently employed to remove the retained material from the column. Alternatively, the analyte can be purified by applying the sample to the column under mobile phase conditions that elute the analyte of interest at a rate that is different from one or more other materials. Such a procedure can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0120] In a preferred embodiment, HTLC can be followed by HPLC on a hydrophobic column chromatography system. In certain preferred embodiments, a TurboFlow Cyclone from Cohesive Technologies is used. Polymer-based columns (60 μm particle size, 50 × 1.0 mm column dimensions, In a related preferred embodiment, a Synergi® from Phenomenex Inc. with a hydrophilic end cap is used. Ether cross-linked phenyl analytical column (4 μm particle size, 150 × 2.0 mm column size, In certain preferred embodiments, HTLC and HPLC are performed using HPLC grade ultrapure water and 100% methanol as mobile phases.

[0121] By carefully selecting valves and connector tubing, two or more chromatographic columns can be connected as needed so that material is passed from one to the next without any manual steps. In a preferred embodiment, the selection of valves and tubing is controlled by a computer preprogrammed to perform the necessary steps. Most preferably, the chromatographic system is also connected to a detector system, e.g., an MS system, in such an online manner. Thus, an operator can place a tray of samples in an autosampler and perform the remaining operations under computer control, which results in purification and analysis of all selected samples.

[0122] In certain preferred embodiments, the amyloid beta or fragments thereof may be purified from the sample prior to ionization. In particularly preferred embodiments, the chromatography method is not gas chromatography.

[0123] Detection and quantification by mass spectrometry

[0124] In a plurality of embodiments, amyloid beta or its fragment can be ionized by any method known to the technician. Use a mass spectrometer to carry out mass spectrometry, which includes an ion source for ionizing fractionated samples and producing charged molecules for further analysis. For example, the ionization of the sample can be carried out by electronic ionization, chemical ionization, electrospray ionization (ESI), proton ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The technician will understand that the selection of ionization method can be determined based on the type of analyte to be measured, the type of sample, the type of detector, the selection of positive to negative mode, etc.

[0125] In a preferred embodiment, amyloid beta or fragments thereof are ionized by heated electrospray ionization (HESI) in positive or negative mode. In an alternative embodiment, amyloid beta or fragments thereof are ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in positive or negative mode.

[0126] After the sample has been ionized, the positively or negatively charged ions thus produced can be analyzed to determine the mass-to-charge ratio. Suitable analyzers for determining the mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Several detection modes can be used to detect ions. For example, that is, the selected ions can be detected using a selective ion monitoring mode (SIM), or alternatively, a scanning mode, such as, multiple reaction monitoring (MRM) or selected reaction monitoring (SRM) can be used to detect ions. Preferably, a quadrupole analyzer is used to determine the mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential, the amplitude of the RF signal, and the mass / charge ratio applied between the electrodes. Voltage and amplitude can be selected so that only ions with a specific mass / charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, a quadrupole instrument can serve as both a "mass filter" and a "mass detector" for the ions injected into the instrument.

[0127] The resolution of MS techniques can be enhanced by adopting "tandem mass spectrometry" or "MS / MS". In this technique, the precursor ions (also referred to as parent ions) generated by the molecule of interest can be filtered in the MS instrument, and the precursor ions are subsequently fragmented to produce one or more fragment ions (also referred to as daughter ions or product ions), which are then analyzed in a second MS program. By carefully selecting the precursor ions, only the ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce fragment ions. Because both the precursor ions and the fragment ions are produced in a reproducible manner under a given set of ionization / fragmentation conditions, the MS / MS technique can provide an extremely powerful analytical tool. For example, the combination of filtering / fragmentation can be used to eliminate interfering substances, and can be particularly useful in complex samples such as biological samples.

[0128] Mass spectrometers usually provide users with ion scans; that is, the relative abundance of each ion with a specific mass / charge within a given range (e.g., 100 to 1000 amu). The results of the analytical test, i.e., mass spectrometry, can be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given careful control of sampling and analysis parameters, the relative abundance of a given ion can be compared with a table that converts the relative abundance into the absolute amount of the original molecule. Alternatively, molecular standards can be run with the sample, and a standard curve is constructed based on the ions generated by those standards. Using such a standard curve, the relative abundance of a given ion can be converted into the absolute amount of the original molecule. In some preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of amyloid protein β. The method of generating and using such a standard curve is well known in the art and a person of ordinary skill can select an appropriate internal standard. For example, an isotope of amyloid protein β can be used as an internal standard. Numerous other methods for correlating the amount of ions with the amount of the original molecule will be well known to a person of ordinary skill in the art.

[0129] One or more steps of the method may be performed using automated machinery.In certain embodiments, one or more purification steps are performed online, and more preferably, all purification and mass spectrometry steps may be performed in an online manner.

[0130] In certain embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, collisionally activated dissociation is often used to generate fragment ions for further detection. In CAD, the precursor ions gain energy by colliding with an inert gas and are subsequently fragmented by a process known as "unimolecular dissociation." Sufficient energy must be accumulated in the precursor ions so that certain bonds within the ions can be broken due to the increased vibrational energy.

[0131] In a particularly preferred embodiment, MS / MS is used to detect and / or quantify amyloid β as follows. The sample is subjected to liquid chromatography, preferably HPLC, and the flow of the liquid solvent from the chromatographic column enters the heated nebulizer interface of the MS / MS analyzer, and the solvent / analyte mixture is converted into steam in the heated charged piping system of the interface. The analyte is ionized by the selected nebulizer. Ions such as precursor ions pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow ions (i.e., "pioneer" ions and "fragment" ions) to be selected based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is a collision cell, in which ions are fragmented. The first quadrupole (Q1) of the mass spectrometer is selected for molecules with the mass-to-charge ratio of amyloid β. Precursor ions with the correct mass / charge ratio of amyloid β are allowed to pass into the collision cell (Q2), while unwanted ions with any other mass / charge ratio collide with the side of the quadrupole and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and fragment. This process is called collision activated dissociation (CAD). The resulting fragment ions are passed to quadrupole 3 (Q3), where the fragment ions of amyloid beta are selected while other ions are eliminated.

[0132] The method may comprise MS / MS performed in positive or negative ion mode.Using standard methods well known in the art, one of ordinary skill will be able to identify one or more fragment ions of a particular precursor ion of amyloid beta that can be used for selection in quadrupole 3 (Q3).

[0133] If the precursor ion of amyloid beta includes an alcohol or amine group, fragment ions are generally formed that represent the dehydration or deamination of the precursor ion, respectively. In the case of a precursor ion including an alcohol group, such fragment ions formed by dehydration are caused by the loss of one or more water molecules from the precursor ion (i.e., wherein the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 18 for the loss of one water molecule, or about 36 for the loss of two water molecules, etc.). In the case of a precursor ion including an amine group, such fragment ions formed by deamination are caused by the loss of one or more ammonia molecules (i.e., wherein the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 17 for the loss of one ammonia molecule, or about 34 for the loss of two ammonia molecules, etc.). Similarly, the precursor ion including one or more alcohol and amine groups generally forms fragment ions representing the loss of one or more water molecules and / or one or more ammonia molecules (i.e., wherein the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 35 for the loss of one water molecule and the loss of one ammonia molecule). Generally, fragment ions representing the dehydration or deamination of the precursor ion are not specific fragment ions for a specific analyte. Therefore, in a preferred embodiment of the present invention, MS / MS is performed in order to detect at least one fragment ion of amyloid beta which does not merely represent the loss of one or more water molecules and / or the loss of one or more ammonia molecules from the precursor ion.

[0134] When ions collide with the detector, they produce electronic pulses that are converted to digital signals. The data obtained are relayed to a computer, which plots the count versus time graph of the collected ions. The mass chromatogram obtained is similar to the chromatogram generated in the traditional HPLC method. The area under the peak corresponding to a specific ion or the amplitude of such a peak is measured, and the area or amplitude are associated with the amount of the analyte of interest. In some embodiments, the area under the curve of fragment ions (one or more) and / or precursor ions or the amplitude at the peak are measured to measure the amount of amyloid beta. As described above, based on the peak of one or more ions of an internal standard molecule, using a calibration standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original analyte.

[0135] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the methods.

[0136] Example

[0137] Example 1 : Device pretreatment and amyloid β stabilization

[0138] Pretreatment of 96-well polypropylene plates and microcentrifuge tubes

[0139] E. coli cells were lysed and precipitated with methanol in a 96-well plate.

[0140] The methanol was then discarded and the pellet was resuspended in ammonium bicarbonate pH 8.

[0141] Sigma bovine trypsin was added and samples were incubated at 60°C for 2 days.

[0142] All digests are then discarded as waste or combined to pre-treat the pipette tip.

[0143] Dry the blank plate and test tubes completely.

[0144] Pretreatment of the pipette tip

[0145] The pipette tip was used to pipette the E. coli digest up and down at least 3 times and then incubated at 60°C for 2 days or until completely dry.

[0146] Amyloid β peptide stabilization

[0147] AB40, AB42, and internal standards were synthesized in pretreated tubes and resuspended in 6 M urea with 4 mg BSA.

[0148] Standards were then diluted 1:10 in 0.1 M PBS using N-terminal and C-terminal antibodies and apolipoprotein E2 and E4, respectively.

[0149] Each mixture was incubated on a shaker at room temperature for 1 hour.

[0150] AB40 and AB42 were then combined and diluted in 0.1 M PBS and 0.4 mg / mL BSA to make high calibrator standards before freezing.

[0151] The table below shows a side-by-side comparison of the same samples prepared in treated and untreated vials.

[0152] AB42 Calculated value processed Unprocessed calculated value % Recovery AB212 2.52 0.33 13.10 AB213 2.14 0.81 37.85 AB214 5.73 1.45 25.31 AB215 0.92 0.21 22.83 AB216 1.85 0.99 53.51 AB40 Calculated value processed Unprocessed calculated value AB212 15.01 2.73 18.19 AB213 13.11 4.93 37.60 AB214 19.18 5.62 29.30 AB215 6.02 2.23 37.04 AB216 7.08 4.76 67.23

[0153] Example 2 : Sample preparation

[0154] Use a pretreated pipette tip to pipette 0.5 mL of sample or standard into the pretreated 96-well plate.

[0155] 5 ng of internal standard was added.

[0156] Add 250 μL of 18 M urea.

[0157] 2 μg of Lys-C was added to each sample for digestion of amyloid β.

[0158] The plates were sealed with adhesive covers before digestion in an enzymatic microwave at 450 w, 45° C. for 4 hours.

[0159] The samples were then extracted using a Waters mixed-mode strong anion exchange.

[0160] The sample eluate was then completely dried using heated nitrogen.

[0161] The samples were then resuspended in reconstitution buffer for LC-MS / MS analysis.

[0162] Example 3 : Detection and quantification of amyloid β fragments by MS / MS

[0163] The ions are passed to the first quadrupole (Q1), which selects ions with a mass-to-charge ratio of 1085.6±0.5 m / z or 1269.7±.5 m / z for Aβ40 fragments and Aβ42 fragments, respectively. The ions entering quadrupole 2 (Q2) collide with argon gas to generate ion fragments, which are passed to quadrupole 3 (Q3) for further selection. At the same time, the same process using isotope dilution mass spectrometry is performed with an internal standard. The following mass transitions are used for detection and quantification during verification of positive polarity.

[0164] Table 1. Amyloid proteins β Mass transition of fragments (positive polarity)

[0165]

[0166] Figure 6 and 7 Example patient chromatograms are shown in .

[0167] Comparison of multiple extractions to optimize recovery of amyloid β fragments.

[0168] Table 2. Comparison of solid phase extraction recovery

[0169]

[0170]

[0171]

[0172] Waters MAX :Mixed-mode strong anion exchange → for final test validation

[0173] Waters MXC : Mixed Mode Strong Cation Exchange

[0174] Waters HLB :Silica for making hydrophobic compounds

[0175] Agilent C18 : Typical C18 filling

[0176] Phenomenex CX : Mixed Mode Strong Cation Exchange

[0177] Thermo SAX : Strong anion exchange

[0178] Mixed mode strong anion exchange provided the best recovery of amyloid β fragments.

[0179] Urea increases the recovery of analytes in digestion. The following values ​​were determined:

[0180] Table 3. Comparison of recovery of urea vs. no urea

[0181]

[0182] The C4 analytical column was compared to the C8 analytical column with respect to sensitivity. The C4 column provided superior sensitivity, which was unexpected since C8 and C18 columns are generally preferred in the art. Fig.11 .

[0183] Example 4 : Test reportable range and linearity

[0184] To establish the linearity of amyloid beta detection in the assay, one blank and spiked standard designated as the zero standard were prepared and analyzed. Quadratic regression from five consecutive runs yielded correlation coefficients of 0.995 or greater with an accuracy of ±20% revealing a quantifiable linear range of 0.1 to 25 ng / mL. Figure 4 and 5 .

[0185] Example 5 : Test accuracy

[0186] The reproducibility of the samples within the assay was tested for the following quality control levels:

[0187] Quality Control Level β-amyloid 40 (pg / mL) β-amyloid 42 (pg / mL) Low 750 750 middle 7500 7500 high 15000 15000

[0188] Within run precision: ten replicates of each quality control were analyzed within a single experiment in the following order; low, medium, and high.

[0189] Acceptance criteria: %CV should be less than the tolerance value ≤ TEa / 2. TEa for this test was determined to be 30%.

[0190] The %CV of amyloid-β 40 ranged from 14.31% to 6.55% for all three quality control levels.

[0191] The %CV for amyloid-β 42 ranged from 14.93% to 7.51% for all three quality control levels.

[0192] Overall precision: Analyze the data from inter-assay validation.

[0193] Acceptance criteria: unacceptable if total SD ≥ 1 / 2TEa, or total SD must be less than the defined maximum SD or CV.

[0194] The %CV should be less than the tolerance value ≤ TEa / 2. The TEa for this test was determined to be 30%.

[0195] The %CV of amyloid beta 40 ranged from 10.21% to 4.28% for all three quality control levels.

[0196] The %CV for amyloid-β 42 ranged from 14.63% to 8.17% for all three quality control levels.

[0197] Example 6 : Analytical sensitivity (detection limit)

[0198] Limit of Blank (LOB): Calculated value: LOB = Mean value of blank + 2SD.

[0199] Twenty-one matrix blanks were analyzed within a single experiment and the back-calculated values ​​were then used to calculate the following LOBs.

[0200] LOB of AB40: 31.32 pg / mL.

[0201] LOB of AB42: 32.11 pg / mL.

[0202]

[0203]

[0204] Limit of Detection (LOD): Calculated value: LOD = Mean of blank + 4SD.

[0205] Twenty-one matrix blanks were analyzed within a single experiment and the back-calculated values ​​were then used to calculate the following LODs.

[0206] LOD of AB40: 77.78 pg / mL.

[0207] LOD of AB42: 74.21 pg / mL.

[0208]

[0209]

[0210] Limit of Quantitation (LOQ): Acceptance Criteria: Lowest concentration with %CV less than or equal to 2SD when TEa is 30%.

[0211] The limit of quantification for both amyloid-β 40 and 42 was determined to be 100 pg / mL.

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] Example 7 : Analyte Measurement Range (AMR)

[0218] Acceptance criteria: When TEa is 30%, the mean of the observed values ​​should deviate from the expected range by no more than 2SD or 20% CV.

[0219] The %CV for amyloid beta 40 ranged from 6.52 to 12.01%, which was considered acceptable.

[0220] The %CV for beta amyloid 42 ranged from 6.27 to 14.24%, which was considered acceptable.

[0221]

[0222]

[0223] Example 8 : Accuracy

[0224] Recovery of known standards.

[0225] Acceptance criteria: When TEa is 30%, the error due to lack of perfect recovery (amount recovered minus amount added) should be ≤ 2SD or 15% CV.

[0226] Six stripped bovine CSF samples were spiked at the following concentrations: 1000, 2000, 3000, 4000, 8000, and 9000 pg / mL, and each spiking level was tested in triplicate.

[0227] Six different spiking levels were then diluted at 1 / 3 and 1 / 5 ratios using treated bovine cerebrospinal fluid as diluent and also tested in triplicate.

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] Example 9 : Sample stability

[0242] Acceptance Criteria: Samples are considered stable if the mean difference between the baseline value and the time / temperature sample value is ≤ TEa / 2 for that analyte when TEa equals 30%.

[0243] Freeze / thaw stability: Freeze-thaw analysis was performed by analyzing six patient pools divided into four equal aliquots. All four aliquots from each patient pool were frozen at -60 to -80°C. Aliquots two to four were thawed to an ambient temperature of 18-26°C and frozen. Aliquots three and four were thawed to an ambient temperature of 18-26°C and frozen. Aliquot four was then thawed to an ambient temperature of 18-26°C and frozen.

[0244] All aliquots were thawed a final time to ambient temperature 18-26°C and were performed in technical triplicate. Freeze-thaw analysis contained data spanning three freeze-thaw cycles.

[0245] Beta-amyloid 40 and beta-amyloid 42 had acceptable stability up to two freeze-thaw cycles.

[0246]

[0247]

[0248] Extracted sample stability: Ten samples were analyzed for baseline values ​​on the same day as sample extraction. The next day, the same samples were injected again for analysis against baseline values.

[0249] Beta-amyloid 40 and 42 showed extracted sample stability up to 1 day at 4°C in the C-stack of the CTC autosampler.

[0250] β Amyloid 40 pg / mL Baseline 1 day average value SD %CV % Recovery Sample 1 0.10 0.11 0.11 0.01 6.73％ 110.00％ Sample 2 0.21 0.22 0.22 0.01 3.29％ 104.76％ Sample 3 0.49 0.52 0.51 0.02 4.20％ 106.12％ Sample 4 1.17 1.07 1.12 0.07 6.31％ 91.45％ Sample 5 2.90 2.86 2.88 0.03 0.98％ 98.62％ Sample 6 4.62 5.20 4.91 0.41 8.35％ 112.55％ Sample 7 25.07 25.37 25.22 0.21 0.84％ 101.20％ Sample 8 0.80 0.76 0.78 0.03 3.63％ 95.00％ Sample 9 8.17 8.02 8.10 0.11 1.31％ 98.16％ Sample 10 16.24 14.33 15.29 1.35 8.84％ 88.24％

[0251] Refrigerated Stability (2.0 to 8.0°C): Samples have been shown to be refrigerated stable for up to 5 days.

[0252]

[0253]

[0254]

[0255]

[0256] Room Temperature Stability (18.0 to 26.0°C): Samples are stable at 18-26°C for up to 3 days.

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] Freeze Stability (-10.0 to -30.0°C): Samples are stable at -10 to -30°C for up to 31 days.

[0263]

[0264]

[0265]

[0266]

[0267] Example 10 : Interference studies

[0268] Acceptance criteria: Differences due to potential interfering substances should be ≤ 2SD or 20% CV to be considered acceptable.

[0269] Hemolytic interference: Six spiked pools were analyzed in triplicate for baseline, mild, moderate, and gross hemolytic interference.

[0270] Beta-amyloid 40 and beta-amyloid 42 are acceptable only for non-hemolyzed and mildly hemolyzed CSF samples.

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278] Lipidemic interference: Six spiked pools were analyzed in triplicate for baseline, mild, moderate, and severe lipidemic interference.

[0279] Beta-amyloid 40 and beta-amyloid 42 are acceptable for mildly and moderately lipidemic CSF samples.

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] Bilirubin Interference: Six spiked pools were analyzed in triplicate for baseline, mild, moderate, and severe icteric interference.

[0287] Beta-amyloid 40 and beta-amyloid 42 are acceptable for anicteric and mildly icteric spinal fluid samples.

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296] Embodiment 11 : Ion suppression

[0297] Ten patient samples were extracted.

[0298] The acquisition window was opened up to 10 minutes to monitor ion suppression throughout the gradient.Ten samples were injected through the analytical column while the digested peptide mixture of amyloid-β 40 and 42 was perfused post-column.

[0299] Ion suppression will be determined to be present in the assay if the total ion chromatogram (TIC) of AB40 or AB42 shows a > 15% decrease in signal intensity when the internal standard of AB40 or AB42 is eluted.

[0300] The TIC of digested peptides of AB40 and 42 showed no suppression in the gradient when the analyte was eluting. The TIC signal intensity was a flat line and showed ≤ 15% signal intensity difference, which is within the acceptable parameters of the assay.

[0301] Example 12 : Carryover

[0302] The high calibrator standard was analyzed followed by four matrix blanks and this sequence was repeated two more times. The average calculated concentration of the high calibrator followed by the matrix blanks yielded a % recovery of 0.06% for both beta amyloid 40 and 42.

[0303] There was no observed carryover for this trial.

[0304]

[0305]

[0306] Embodiment 13 : Reference interval (RI)

[0307] β-amyloid protein 40: 6000.00-15000.00pg / mL

[0308] β-amyloid 42: 700.00-4000.00 pg / mL

[0309] Embodiment 14 : Alzheimer's disease patient data

[0310] Cerebrospinal fluid (CSF) of 211 subjects, including patients diagnosed with Alzheimer's disease and normal subjects, were analyzed. Figure 8-10 .

[0311] Aβ40 and Aβ42 were detected in all CSF samples. Surprisingly, Aβ42 levels were about 10 times higher than those that have been published. Most patient samples were in the range of 1 to 8 ng / mL. Alzheimer's patients were distinguishable based on the low ratio of Aβ42:Aβ40 compared to borderline patients and normal subjects (highest ratio). Figure 8 .

[0312] Embodiment 15 : Additional recovery studies

[0313] Sample preparation: All plastic disposables were pretreated and Aβ standards were stabilized to prevent nonspecific binding and enhance long-term storage stability.

[0314] Strong protein denaturants were added to human CSF (500 μL) and samples were subjected to protein digestion followed by solid phase extraction.Samples were processed on a robotic liquid handler (Hamilton MicrolabStar A857) connected to a CEREX IP8 (SPEware) solid phase extraction manifold.

[0315] Separation: On a Waters XBridge Protein BEH C4 column, 4.6 × 100 mm, 3.5 μm HPLC separations were performed on an Aria TLX-4 system (Thermo Scientific).

[0316] Detection: Thermo TSQ Quantiva triple quadrupole mass spectrometer.

[0317] Linearity data showed R values ​​of at least 0.98 for both peptides. 2 Values ​​were obtained with %CV ≤ 15% across the 8 calibrator standards.

[0318] The limit of quantification for both peptides was 100 pg / mL.

[0319] The assay precision (%CV) was ≤15%, with recoveries of Aβ40 and Aβ42 ranging from 84% to 112%.

[0320] Intra-experimental (N=10) and inter-experimental (N=5, 5 days) precision and accuracy

[0321]

[0322]

[0323] Test stability within 8 months

[0324]

[0325] Stabilization of Aβ peptides is achieved, which allows long-term storage of calibrators and quality control standards.

[0326] When stored at -80°C, the frozen calibrators are stable for at least 8 months.

[0327] Stabilization measures eliminate nonspecific binding and result in higher analyte recovery. Fig.12 and 13 .

[0328] The values ​​of CSF Aβ42 concentrations in patients using the LC-MS / MS assay were higher compared to those using the ELISA assay. This is consistent with previous reports comparing the 2 methods. Further work will determine whether the preanalytical factors described here can help explain the differences.

[0329] patient LC-MS / MS (pg / mL) ELISA (pg / mL) 1 1,668 536 2 1,617 534 3 2,808 624 4 3,184 850 5 4,061 913 6 2,142 672 7 1,878 627 8 2,829 566 9 1,882 633 10 1,634 614 11 2,706 644 12 773 466 13 1,801 602 14 3,891 739 15 1,524 671 16 2,300 605 17 1,167 458 18 1,842 625 19 2,431 665 20 2,814 938

[0330] Conclusion: This novel method eliminates one of the major challenges in quantifying Aβ40 and Aβ42: poor reproducibility caused by nonspecific binding.

[0331] Example 16 : Patient Diagnosis Research

[0332] Three quality controls (low, medium, and high) were run, representing three different points along the calibration curve. QCs were run at the beginning and end of the run to ensure accurate quantification across the plate. Quality control accuracy is shown below:

[0333] Aβ40 Expected value (pg / mL) Calculated value (pg / mL) % Accuracy Before: Low QC 750 743.28 99.10％ Front: Middle QC 7500 6724.33 89.66％ Front: High QC 15000 13554.62 90.36％ After: Low QC 750 754.58 100.61％ After: Middle QC 7500 7351.87 98.02％ After: High QC 15000 13911.65 92.74％

[0334] Aβ42 Expected value (pg / mL) Calculated value (pg / mL) % Accuracy Before: Low QC 750 815.71 108.76％ Front: Middle QC 7500 6930.69 92.41％ Front: High QC 15000 13976.57 93.18％ After: Low QC 750 667.22 88.96％ After: Middle QC 7500 6341.3 84.55％ After: High QC 15000 13384.45 89.23％

[0335] Aβ40 and 42 patient samples range

[0336] Based on 72 patient samples

[0337] Aβ40: 5135.33 - 25348.94 pg / mL

[0338] Aβ42: 1068.00 - 5499.76 pg / mL

[0339] Normalized change levels of Aβ40 and 42.

[0340] As the Aβ42 value decreases due to plaqueing or inadequate clearance, the ratio of Aβ42 / 40 also decreases.

[0341] Aβ40 and 42 levels show independent increases / decreases from each other.

[0342] Data was classified by the Aβ42 / 40 ratio and divided into two groups based on the median (Aβ42 / 40 = 0.17)

[0343] Half of the data went into < median of Aβ42 / 40

[0344] Half of the data went into > median of Aβ42 / 40

[0345] The mean, standard deviation, and %CV were calculated:

[0346] For the ratio < median of Aβ42 / 40, values ≥ 1SD were removed

[0347] For the ratio > median of Aβ42 / 40, values ≤ 1SD were removed

[0348] 1SD outliers were placed into the "critical" category

[0349] The content of articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, is hereby incorporated by reference in its entirety, as if each individual publication was specifically and individually indicated to be incorporated by reference. The applicant reserves the right to physically incorporate any and all materials and information from any such article, patent, patent application, or other physical and electronic documents into this application.

[0350] The methods illustratively described herein may be suitable for practice in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be understood broadly and non-restrictively. In addition, the terms and expressions adopted herein have been used as descriptive and non-restrictive terms, and it is not intended to use such terms and expressions that exclude any equivalents of the features shown and described, or portions thereof. It should be recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the present invention embodied therein as disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0351] The present invention has been described broadly and generally herein. Each of the narrower classes and subgroups falling within the general disclosure also forms part of the method. This includes the use of restrictive clauses or negative limitations to the general description of the method, which removes any subject matter from the category, regardless of whether the removed material is specifically cited herein.

[0352] Other embodiments are within the scope of the following claims.In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method for determining the amount of amyloid β in a sample for non-therapeutic and non-diagnostic purposes, the method comprising: (a) pre-treating the surface of the device in contact with the sample with an agent that prevents the amyloid β fragments from adhering to the surface; (b) digesting amyloid β in the sample to produce one or more fragments of amyloid β, wherein the one or more fragments include amyloid β 40 (Aβ40) comprising the sequence GAIIGLMVGGVV (SEQ ID NO: 2) or amyloid β 42 (Aβ42) comprising the sequence GAIIGLMVGGVVIA (SEQ ID NO: 4); (c) purifying the one or more amyloid β fragments; (d) ionizing the amyloid β fragments to generate precursor ions; and (e) generating one or more fragment ions; (f) determining the amount of said ion(s) from step (d) or (e) or both (d) and (e) by mass spectrometry; wherein the amount of said ion is correlated to the amount of one or more amyloid beta fragments in said sample, and (g) determining the ratio of Aβ42 to Aβ40; and The reagent is E. coli lysate.

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

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

4. The method of claim 1, wherein the method further comprises a C-4 analytical column.

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

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

7. The method of claim 5, wherein the agent confers stability through at least three freeze-thaw cycles.

8. The method of claim 5, wherein the agent confers stability at -70°C for at least 2 months.

9. The method of claim 1, wherein the method comprises mixed mode anion exchange extraction.

10. The method of claim 1, wherein the ionizing comprises heated electrospray ionization (HESI).

11. The method of claim 1, wherein the ionizing comprises ionizing in a positive mode.

12. The method of claim 1, wherein generating fragment ions comprises using a collision energy between 20 V and 45 V.

13. The method according to claim 1, wherein the mass-to-charge ratio of the precursor ion is 1085.6±0.5 or 1269.7±0.

5.

14. The method of claim 1, wherein the mass / charge ratio of the one or more fragment ions is 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.

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

16. The method of claim 15, wherein the internal standard is isotopically labeled.

17. The method of claim 16, wherein the internal standard comprises 13 C 15 N mark.

18. The method of claim 16, wherein the precursor ion of the internal standard has a mass / charge ratio of 1110.7±0.

5.

19. The method of claim 16, 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.

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

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

22. The method of claim 1, wherein the digestion comprises digestion with Lys C.

23. The method of claim 22, wherein the digesting further comprises adding urea.

24. The method of claim 22, wherein the digesting further comprises digesting in a microwave.

25. The method of claim 1, wherein the amyloid beta fragments further comprise a wing peptide.

26. The method of claim 25, wherein the winged peptide is hydrophilic.

27. The method of claim 25, wherein the winged peptide comprises one or more N-terminal or C-terminal amino acid residues.

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