Method for mass spectrometric quantification of amyloid beta peptides in plasma

Abstract

The present invention relates to a method for preparing a plasma sample comprising amyloid beta peptides for analysis by mass spectrometry, comprising the steps of: a) contacting the plasma sample with a denaturing agent; b) performing a first solid-phase extraction step on the solution obtained in step a) to recover a first eluate; c) performing a second solid-phase extraction step on the first eluate obtained in step b) to recover a second eluate; and d) drying the second eluate obtained in step c) and processing it for analysis by mass spectrometry, wherein the solution obtained in step d) comprises intact amyloid beta peptides Aβ40 and Aβ42. JPEG2023523203000005.jpg71170

Description

[Technical Field]

[0001] The present invention relates to the field of human and veterinary medicine in the diagnosis of amyloid diseases, and in particular to analytical methods for the preparation of plasma samples for the detection and quantification by mass spectrometry of amyloid Aβ40 and Aβ42 peptides. [Background technology]

[0002] Alzheimer's disease (AD), the leading cause of dementia, is characterized by a progressive degenerative disorder of the central nervous system, affecting 17% of people aged 75–84 and 32% of people over 85 (Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, January 2020, pp. 194–208). AD is characterized by the progressive appearance in patients' brains of amyloid plaques, which have a central core of amyloid deposits formed primarily by fibrils of 40–42 amino acid peptides. These amyloid deposits form following proteolytic processing of the amyloid precursor protein (APP), resulting in the generation of insoluble Aβ peptides, predominantly Aβ1–40 (Aβ40) and Aβ1–42 (Aβ42). In healthy patients, these peptides are either cleared and enter the cerebrospinal fluid (CSF) or transported across the blood-brain barrier into the blood. However, overproduction or reduced clearance of amyloid peptides leads to the formation of amyloid plaques characteristic of AD. These plaques contain primarily Aβ42 and act as a "sink" for the peptide, thereby reducing Aβ42 concentrations in both CSF and blood.

[0003] Therefore, the Aβ42 / Aβ40 concentration ratio is currently used as a cerebral amyloidosis biomarker in the early stages of Alzheimer's disease, which is included in clinical trials. Aβ42 and Aβ40 can be measured by either mass spectrometry (MS) or immunoassay, and most currently available methods are performed in cerebrospinal fluid (CSF), where reduced Aβ42 concentrations are observed in the presence of amyloid plaques (Bateman and Budelier, Biomarkers of Alzheimer's Disease, Journal of Applied Laboratory Medicine, January 2020, pp. 194-208). However, CSF collection is quite invasive, requires specialized medical skills, and is not the most convenient method for large-scale screening studies. Therefore, methods for quantifying Aβ42 and Aβ40 in plasma are of great interest in the diagnosis of symptomatic and asymptomatic individuals (Fandos et al., Plasma amyloid β 42 / 40 ratios as biomarkers for amyloid β cerebral deposition in cognitively normal individuals. Alzheimer's Dement, 2017 Sep 12; 8:179-187).

[0004] Nevertheless, measuring Aβ42 and Aβ40 concentrations in plasma presents challenges. First, blood is a highly complex matrix containing a wide variety of proteins, with the total protein content in plasma being 60 times higher than that in CSF. Second, the concentrations of Aβ42 and Aβ40 are lower than those in CSF because they are transported from the central nervous system into venous blood. Furthermore, the difference in Aβ42 concentrations between amyloid-positive and amyloid-negative individuals in plasma is smaller than in CSF (Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, January 2020, pp. 194-208). For these reasons, analyzing amyloid beta peptides in plasma is more challenging than in CSF, and therefore, more sensitive and accurate methods are needed.

[0005] Previous studies using enzyme-linked immunosorbent assays (ELISAs) to measure plasma amyloid beta peptide concentrations have shown conflicting results regarding Aβ42 / Aβ40 concentration ratio measurements in AD patients and healthy controls (Fukumoto et al., "Age but not diagnosis is the main predictor of plasma amyloid beta-protein levels," Arch Neurol. 2003;60:958–964; Perez-Grijalba et al., "Plasma Aβ42 / 40 ratio alone or combined with FDG-PET can accurately predict amyloid-PET positivity: a cross-sectional analysis from the AB255 Study," Alzheimer's Res Ther. 2019;11:96). Indeed, the mean difference between groups (i.e., between healthy controls and individuals with mild cognitive impairment) was low, at 10–15%, which is within the acceptable variability range for the accuracy and precision of analytical methods. This suggests that the variability of the analytical method would have to be much less than 15% to detect this small difference between groups.

[0006] On the other hand, available mass spectrometry (MS) methods have demonstrated greater sensitivity and accuracy than immunoassays. Currently, two MS-based analytical methods for the determination of Aβ40 and Aβ42 in human plasma are known in the art. The first method was published in 2017 by Randall Bateman's group (Ovod et al., Amyloid B concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis, Alzheimer's and Dementia, October 2017; 13(10): 1185). This method combines sample preparation by immunoprecipitation and Lys-N digestion with nanoLC-MS / MS. The second method, published in 2018 by Akinori Nakamura's group (Nakamura et al., High performance plasma amyloid-β biomarkers for Alzheimer's disease, Nature 2018 Feb 8;554(7691):249-254), combines sample preparation by double immunoprecipitation with MALDI-TOF / MS.

[0007] However, both methods are time- and resource-consuming, as they require the use of expensive antibodies during immunoprecipitation. Furthermore, Bateman's method requires additional analyte enzymatic digestion of the sample, which results in the detection of a mixture of N-cleaved Aβ species rather than intact Aβ peptides. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, January 2020, pp. 194-208 [Non-patent document 2] Fandos et al., Plasma amyloid β 42 / 40 ratios as biomarkers for amyloid β cerebral deposition in cognitively normal individuals. Alzheimer's Dement, September 12, 2017; 8:179–187 [Non-patent document 3] Fukumoto et al., Age but not diagnosis is the main predictor of plasma amyloid beta-protein levels, Arch Neurol. 2003; 60:958-964 [Non-patent document 4] Perez-Grijalba et al., Plasma Aβ42 / 40 ratio alone or combined with FDG-PET can accurately predict amyloid-PET positivity: a cross-sectional analysis from the AB255 Study, Alzheimer's Res Ther. 2019; 11:96 [Non-Patent Document 5] Ovod et al., Amyloid B concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis, Alzheimer's and Dementia, October 2017; 13(10): 1185 [Non-patent document 6] Nakamura et al., High performance plasma amyloid-β biomarkers for Alzheimer's disease, Nature 2018;2;554(7691):249-254 Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there remains a need in the art for a sensitive and reproducible method for the detection and quantification of amyloid beta peptide in plasma samples that is applicable to large-scale screening studies. [Means for solving the problem]

[0010] After extensive and thorough experimentation, the inventors of the present invention have surprisingly discovered a novel method for the preparation of plasma samples containing amyloid peptides that allows accurate quantification of intact amyloid peptides Aβ40 and Aβ42 by mass spectrometry. Thus, the novel method reduces the variability of the measured Aβ42 / Aβ40 ratio to a value that is smaller than the actual difference between healthy controls and individuals with mild cognitive impairment.

[0011] Furthermore, the methods of the present invention are performed without the use of immunoprecipitation or digestion of the sample, which are essential steps in methods known in the art, thus providing simpler and faster sample preparation compared to currently available methods, reducing the cost and time required.

[0012] Finally, the methods of the present invention comply with current FDA recommendations for bioanalytical method validation and are therefore applicable to diagnosing and / or differentiating between different stages of neurodegenerative diseases, such as Alzheimer's disease.

[0013] In one aspect, the present invention provides a method for preparing a plasma sample comprising amyloid beta peptide for analysis by mass spectrometry, comprising the steps of: a) contacting the plasma sample with a denaturing agent; b) subjecting the solution obtained in step a) to a first solid phase extraction step to recover a first eluate; c) subjecting the first eluate obtained in step b) to a second solid phase extraction step to recover a second eluate; and d) drying the second eluate obtained in step c) and processing it for analysis by mass spectrometry; The method is characterized in that the sample obtained from step d) contains intact amyloid beta peptides Aβ40 and Aβ42.

[0014] In one embodiment of the method, the second solid phase extraction step is a cation exchange solid phase extraction.

[0015] In another embodiment, the cation exchange solid phase extraction is strong cation exchange, weak cation exchange, or mixed mode reversed phase cation exchange.

[0016] In another embodiment, the second solid phase extraction step is anion exchange solid phase extraction.

[0017] In another embodiment, the second anion exchange solid phase extraction is strong anion exchange, weak anion exchange, or mixed mode reversed phase anion exchange.

[0018] In another embodiment, contacting the plasma sample with an acidic denaturing agent in step a) results in a solution having a pH of 4.5 or less.

[0019] In another embodiment, the acidic denaturant is an aqueous solution of formic acid at a concentration of 40% to 70% (v / v).

[0020] In another embodiment, the first solid phase extraction step is a reversed phase solid phase extraction.

[0021] In another embodiment, the first and second solid-phase extraction steps each include at least two washing steps, wherein the first washing step of the first and second solid-phase extraction steps is performed with a solution containing an acid, and the second washing step of the first and second solid-phase extraction steps is performed with a solution containing a water-miscible polar organic solvent.

[0022] In another embodiment, the acid-containing solution of the first cleaning step is different from the acid-containing solution of the second cleaning step.

[0023] In another embodiment, the acid-containing solution of the first washing step is the same as the acid-containing solution of the second washing step.

[0024] In another embodiment, the first solid phase extraction step is a cation exchange solid phase extraction.

[0025] In another embodiment, the first cation exchange solid phase extraction is strong cation exchange, weak cation exchange, or mixed mode reversed phase cation exchange.

[0026] In another embodiment, the first and second solid-phase extraction steps each include at least two washing steps, wherein the first washing step of the first solid-phase extraction is performed with a solution containing an acid, the first washing step of the second solid-phase extraction is performed with a solution containing a base, and the second washing step of the first and second solid-phase extraction steps is performed with a solution containing a water-miscible polar organic solvent.

[0027] In another embodiment, contacting the plasma sample with a basic denaturing agent in step a) results in a solution having a pH of about 11 or greater.

[0028] In another embodiment, the basic denaturant is an aqueous solution of ammonium hydroxide at a concentration of 5% to 50% (v / v).

[0029] In another embodiment, the first solid phase extraction step is anion exchange solid phase extraction.

[0030] In another embodiment, the first anion exchange solid phase extraction is strong anion exchange, weak anion exchange, or mixed mode reversed phase anion exchange.

[0031] In another embodiment, the first and second solid-phase extraction steps each include at least two washing steps, wherein the first washing step of the first solid-phase extraction is performed with a solution containing an acid, the first washing step of the second solid-phase extraction is performed with a solution containing a base, and the second washing step of the first and second solid-phase extraction steps is performed with a solution containing a water-miscible polar organic solvent.

[0032] In yet another embodiment, the solution for treating the second eluate for analysis by mass spectrometry is an aqueous solution comprising a surfactant and a reducing agent. In a preferred embodiment, the solution of step d) for treating the dried eluate is an aqueous solution comprising Triton X-100 at a concentration of 0.01% to 0.8% (v / v) and tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v).

[0033] In another embodiment, the solution in step d) for treating the dried eluate is an aqueous solution containing a surfactant, a reducing agent, a water-miscible polar organic solvent, and an acid. In a preferred embodiment, the solution in step d) for treating the dried eluate is an aqueous solution containing Triton X-100 at a concentration of 0.01% to 0.8% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), dimethylformamide at a concentration of 0.1% to 3% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v).

[0034] In some embodiments of the present invention, the plasma sample is a human plasma sample. In other embodiments, the volume of the plasma sample used in step a) of the method of the present invention is between 100 μL and 400 μL.

[0035] In some embodiments of the present invention, the method for preparing a plasma sample comprising amyloid beta peptides for analysis by mass spectrometry does not involve immunoprecipitation or digestion of the plasma sample prior to analysis by mass spectrometry.

[0036] In a second aspect, the present invention provides a method for the mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample, comprising steps a) to d) of the method described herein for preparing the plasma sample, and further comprising: i) subjecting the solution obtained in step d) to a liquid chromatography step to separate the analytes of interest; ii) subjecting the analytes separated in step i) to ionization to produce one or more charged species; iii) separating the one or more charged species according to ion mobility; iv) detecting the one or more charged species separated in step iii) and determining their abundance by mass spectrometry; and v) determining the amount or concentration of intact amyloid beta peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged species measured in step iv) with a standard curve.

[0037] In some embodiments, the method for mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in plasma samples is characterized in that the liquid chromatography is micro-liquid chromatography (micro-HPLC), the ionization is electrospray ionization (ESI), the separation of one or more charged species is performed by differential mobility spectrometry (DMS), and the mass spectrometric technique for detecting and measuring the abundance of the separated one or more charged species is multiple reaction monitoring (MRM) in a triple quadrupole instrument.

[0038] In another embodiment of the invention, the standard curve used in the method for mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in plasma samples is generated with human plasma.

[0039] In a third aspect, the present invention relates to an aqueous solution for treating a dried eluate to be analyzed by mass spectrometry, comprising Triton X-100 at a concentration of 0.01% to 0.8% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), dimethylformamide at a concentration of 0.1% to 3% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v). [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a graph showing the Aβ42 / Aβ40 ratios corresponding to samples from 36 individuals (PET-negative or PET-positive) quantified by the method of the present invention. [Figure 2] 2 is a graph of the ROC curve calculated for the Aβ42 / Aβ40 ratio of FIG. 1. [Figure 3] Chromatograms obtained for plasma samples subjected to mixed-mode reversed-phase cation exchange (MCX) as the only SPE step: the left trace is for Aβ40 and the right trace is for Aβ42. [Figure 4] Chromatogram obtained for a plasma sample subjected to a first SPE step, reversed phase (HLB prime), followed by a second SPE MCX according to protocol A. The left trace relates to Aβ40 and the right trace relates to Aβ42. [Figure 5] Chromatograms obtained for a plasma sample subjected to a first SPE step, MCX, followed by a second SPE, HLB. The left trace is for Aβ40 and the right trace is for Aβ42. [Figure 6]Chromatogram obtained for a plasma sample subjected to a first SPE step, HLB, followed by a second SPE, MCX, according to protocol B. The left trace relates to Aβ40 and the right trace relates to Aβ42. [Figure 7] Chromatograms obtained for plasma samples subjected to a first SPE step, mixed-mode reversed-phase anion exchange SPE (MAX), followed by a second SPE, MCX, according to Protocol D, compared to the combination of a first HLB followed by a second MCX. The left trace is for Aβ40, and the right trace is for Aβ42. [Figure 8] Chromatograms obtained for plasma samples subjected to a first SPE step, MCX, followed by a second SPE MAX according to Protocol C, compared to a first HLB followed by a second MCX combination. The left trace is for Aβ40, and the right trace is for Aβ42. DETAILED DESCRIPTION OF THE INVENTION

[0041] The following description is intended only to illustrate various embodiments of the present invention. Accordingly, the specific modifications described are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, modifications, and variations can be made without departing from the spirit or scope of the subject matter presented herein, and it will be understood that these equivalent embodiments are to be included herein.

[0042] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0043] Throughout this specification and claims, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the stated element or elements but not the exclusion of any other element or elements.

[0044] As used herein, the terms "analyte," "chemical species," "sample," "component," "chemical substance," and "ion" may all be used to refer to substances to be analyzed, identified, and quantified by the methods of the present invention.

[0045] As used herein, the term "solid-phase extraction" or "SPE" refers to a method for separating a mixture into multiple components. The components are dissolved and / or suspended in a solution (the "sample solution") and are separated from one another by their differential affinity for a solid (the "stationary phase") through which the solution is passed. In some cases, undesired components of the sample solution may be retained on the stationary phase as the sample solution passes through it (i.e., the analytes in the sample solution are purified). In other cases, desired components may be retained on the stationary phase (i.e., the analytes of interest are retained on the stationary phase), and a second mobile phase is used to elute the analytes retained on the stationary phase for further processing or analysis. The "stationary phase" is typically held in a "cartridge," "chip," or "column," which can be assembled into multiwell plates, which are particularly convenient for large-scale screening studies. Solid-phase extraction cartridges, columns, chips, and multiwell plates are commercially available or can be prepared according to methods known in the art.

[0046] The term "purification" refers to a procedure that enriches the amount of one or more analytes of interest relative to other components in a sample that may interfere with the detection of the analytes of interest. As used herein, the term "purification" does not refer to the removal of all substances other than the analytes of interest from a sample.

[0047] "Immunoprecipitation" refers to a purification procedure that utilizes antibodies, including polyclonal or monoclonal antibodies, to enrich for one or more analytes of interest in a sample.

[0048] As used herein, the term "digestion" generally refers to any suitable method for breaking down or cleaving polypeptides or proteins, including, for example, the use of cellular enzymes (proteases) and intramolecular digestion.

[0049] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique for measuring the mass-to-charge ratio of a particular analyte. MS is used broadly to include all components and systems that can be used to detect and identify analytes using their mass-to-charge ratio. MS techniques generally involve ionizing the analyte (although the analyte may already be ionized in solution) to form charged analytes, transferring these charged analytes to the gas phase, determining the mass-to-charge ratio, and calculating the relative or absolute abundance. The analytes can be ionized and detected by any suitable means.

[0050] The term "chromatography" refers to a method for separating a liquid- or gas-borne mixture into components that elute at different retention times as a result of differences in the distribution of chemical entities as the components pass through a stationary phase. Thus, "liquid chromatography" (LC) or "high-performance liquid chromatography" (HPLC) refers to the selective separation of one or more components of a fluid solution as the fluid passes through a column. This separation occurs because the components of the mixture are partitioned between one or more stationary phases and a mobile phase. Examples of LC or HPLC include normal-phase liquid chromatography (NPLC), reverse-phase liquid chromatography (RPLC), high-turbulence liquid chromatography (HTLC), hydrophilic interaction chromatography (HILIC), ion-exchange chromatography (IEC), size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), electrostatic repulsion liquid chromatography (ERLIC), and multidimensional liquid chromatography.

[0051] The terms "micro HPLC" or "micro LC" refer to high performance liquid chromatography (HPLC) using a micro flow rate (i.e., 1-25 μl / min) and a capillary column (inner diameter 150-500 μm).

[0052] The terms "ionization" or "ionize" refer to a method of producing analyte ions having a net charge equal to one or more charge units. Anions have a net negative charge and cations have a net positive charge. Non-limiting examples of ionization include electron impact ionization, chemical ionization, electrospray ionization (ESI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI), and atmospheric pressure chemical ionization (APCI), among others.

[0053] "Electrospray ionization" or "ESI" refers to an ionization method in which analytes of interest are transferred into the gas phase as ions and a sample solution containing the analytes of interest is sprayed into an electric field to form charged droplets.

[0054] The term "ion mobility spectrometry (IMS)" refers to an analytical technique used to separate and identify ionized molecules in the gas phase based on their mobility in a carrier buffer gas. The term "differential mobility spectrometry (DMS)" refers to a specific type of IMS that consists of separating ionized molecules based on the difference between their ion mobility in a high electric field and their ion mobility in a low electric field in a gas at or near atmospheric pressure.

[0055] The term "multiple reaction monitoring (MRM)", also known as "selected reaction monitoring (SRM)", refers to a scan mode of tandem MS in which two (or more) analytical devices (i.e., quadrupoles) are tuned to monitor one or more selected parent-product pairs of an analyte of interest.

[0056] The terms "tandem mass spectrometry" or "MS / MS" refer to mass spectrometry involving multiple stages of mass analysis separated in time or space. For example, time-differential tandem mass spectrometry can involve a single mass analyzer (e.g., an ion trap) that first traps, isolates, and fragments specific ions, and then analyzes the fragments in the same analyzer. Spatial-differential tandem mass spectrometry involves two or more analyzers. The analyzers are separated by one or more reaction regions (i.e., collision cells filled with gases such as argon, xenon, nitrogen, or helium) where analyte separation occurs. Finally, the fragment ions are filtered in a final analyzer before detection. Typically, two analyzers are used, which may or may not be the same type.

[0057] As used herein, the term "ROC" refers to "Receiver Operating Characteristics." ROC analysis can be used to evaluate the diagnostic or predictive capabilities of a test or analytical method. An ROC graph is a plot of the sensitivity and specificity of a test at various thresholds or cutoff values. Each point on the ROC curve represents the sensitivity and its respective specificity. A threshold can be selected based on the ROC curve to identify a point where both sensitivity and specificity are acceptable, and this value can be used to apply the test for diagnostic purposes. If specificity alone is optimized, the test will be less likely to produce false positives (an increase in the number of cases of disease diagnosed in subjects without the disease), but at the cost of increasing the likelihood that some disease cases will not be identified (e.g., false negatives). If sensitivity alone is optimized, the test will be more likely to identify most or all subjects with the disease, but will also increase the number of cases of disease diagnosed in subjects without the disease (e.g., false positives). Users can modify the parameters and thus select an ROC threshold suitable for a given clinical condition in a manner readily understood by those skilled in the art.

[0058] The term "area under the curve (AUC) value" quantifies the overall ability of a test to distinguish between different sample characteristics, in this case, between subjects with Aβ amyloidosis (i.e., amyloid-positive) and subjects without Aβ amyloidosis (i.e., amyloid-negative). A test that identifies true positives by chance alone will produce an ROC curve that exhibits an AUC of 0.5. A test that exhibits perfect specificity and sensitivity (i.e., produces no false positives or false negatives) will exhibit an AUC of 1.00.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used and will be apparent to those skilled in the art.

[0060] Unless expressly stated otherwise, each embodiment in this specification applies mutatis mutandis to all other embodiments.

[0061] The present invention relates to a method for preparing a plasma sample containing amyloid beta peptide for analysis by mass spectrometry.

[0062] Amyloid beta peptides (also called Aβ or Abeta peptides) are peptides obtained by the proteolytic process of amyloid precursor protein (APP). As used herein, the term "amyloid beta" refers to total amyloid beta (Aβ) protein, Aβ40, Aβ42, or another Aβ isoform. In some embodiments, the sample may contain Aβ40. In other embodiments, the sample may contain Aβ42. In preferred embodiments, the sample may contain Aβ40 and Aβ42.

[0063] As used herein, the term "intact" amyloid beta refers to a full-length peptide that has not been subjected to chemical / enzymatic cleavage or any other peptide modification. In the case of Aβ40 and Aβ42 peptides, intact amyloid beta peptide Aβ42 refers to a 42-amino acid peptide corresponding to amino acids 672-713 of human APP isoform 770 (canonical, accession number P05067 in UniProtKB), and intact amyloid beta peptide Aβ40 refers to a 40-amino acid peptide corresponding to amino acids 672-711 of human APP isoform 770 (canonical, accession number P05067 in UniProtKB).

[0064] In some embodiments, the sample is a plasma sample from a subject. Suitable subjects include humans or any other mammals, livestock animals (such as pigs, cows, horses, goats, sheep, llamas, and alpacas), companion animals (such as dogs, cats, rabbits, and birds), laboratory animals (such as rodents, e.g., mice, rats, guinea pigs), or zoo animals. In preferred embodiments, the subject is a mammal. In more preferred embodiments, the subject is a human.

[0065] The plasma sample may be used "as is," or a protein fraction may be isolated from the plasma sample using standard techniques. For example, the plasma sample may be concentrated, diluted, or extracted. Suitable extraction techniques may include detergents, acids, bases, organic solvents, or other methods known in the art. In some embodiments, the raw sample may be pre-treated to reduce the complexity of the matrix. In some embodiments, these pre-treatments are techniques well known to those of skill in the art, such as liquid-liquid extraction or protein precipitation, although other techniques known to those of skill in the art are not excluded. In some embodiments, plasma samples are preferred. In a more preferred embodiment, the plasma sample is a human plasma sample.

[0066] In a first aspect, the present invention relates to a method for preparing a plasma sample containing amyloid beta peptide for analysis by mass spectrometry, said method comprising: a) contacting the plasma sample with a denaturing agent; b) subjecting the solution obtained in step a) to a first solid phase extraction step to recover a first eluate; c) subjecting the first eluate obtained in step b) to a second solid phase extraction step to recover a second eluate; and d) drying the second eluate obtained in step c) and processing it for analysis by mass spectrometry; The sample obtained from step d) contains intact amyloid beta peptides Aβ40 and Aβ42.

[0067] The term "processing" in step d) means adapting the sample for analysis by mass spectrometry, including for example adapting the sample for a combination of liquid chromatography and mass spectrometry.

[0068] In some embodiments, the plasma sample containing amyloid beta peptide is contacted with an acidic denaturant. In a more preferred embodiment, the acidic denaturant is an organic acid. In an even more preferred embodiment, the organic acid is a carboxylic acid. In an even more preferred embodiment, the carboxylic acid is a monocarboxylic acid, a dicarboxylic acid, or a tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about 6 or less, for example, about 5 or less, for example, about 4 or less. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about 4 or less. In a preferred embodiment, the monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid is a C1-C10 haloalkyl, a C1-C10 alkyl, or a combination thereof. For example, the carboxylic acid may be selected from a C1-C5 haloalkyl monocarboxylic acid, a C1-C5 alkyl monocarboxylic acid, and a combination thereof. In an even more preferred embodiment, the carboxylic acid is formic acid.

[0069] All pKa values ​​disclosed herein are measured in water at 25° C. and 1 atm pressure.

[0070] Thus, in some embodiments, the acidic denaturant is formic acid. In more preferred embodiments, the acidic denaturant is an aqueous formic acid solution containing 40% to 70% (v / v) formic acid. In preferred embodiments, the formic acid solution contains 45% to 60% (v / v) formic acid in water. In even more preferred embodiments, the formic acid solution contains about 50% (v / v) formic acid in water.

[0071] In some preferred embodiments, a solution having a pH of about 4.5 or less is obtained by contacting a plasma sample containing amyloid beta peptide with an acidic denaturant. In more preferred embodiments, the pH of the solution may be about 0.1 to about 4.5. For example, the pH of the solution may be about 0.4 to about 3. The pH of the solution may be about 0.5 to 2, for example, about 0.8 to 1.5. In more preferred embodiments, the solution obtained after contacting the plasma sample with the acidic denaturant has a pH of 1 to 1.4, more preferably 1.1 to 1.3, and more preferably about 1.2.

[0072] The amount of acidic denaturant used in the method of the present invention depends on the amount of plasma sample used and the pH of the acidic denaturant. Those skilled in the art can determine the amount of acidic denaturant to obtain a solution with a specific pH by simple calculation. In some preferred embodiments of the present invention, the acidic denaturant is an aqueous solution of formic acid with a concentration of 40% to 70% (v / v). In a more preferred embodiment of the present invention, a plasma sample in an amount of 100 μl to 400 μl is contacted with an aqueous solution of formic acid in an amount of 200 μl to 800 μl with a concentration of 40% to 70% (v / v).

[0073] The use of formic acid as a denaturing agent to denature the plasma samples of the present invention provides several advantages, for example, it disrupts many Aβ-plasma protein interactions (i.e., IgG) while keeping the Aβ peptide in solution (no precipitation occurs).

[0074] In some embodiments, the plasma sample containing amyloid beta peptide is contacted with a basic denaturing agent. In more preferred embodiments, the basic denaturing agent is selected from the group consisting of water-soluble hydroxides, carbonates, oxides, and combinations thereof. For example, the base can be a water-soluble hydroxide. Suitable hydroxides include inorganic hydroxides, organic hydroxides, and combinations thereof. In a preferred embodiment, the base is ammonium hydroxide. In a more preferred embodiment, the basic denaturing agent is an aqueous ammonium hydroxide solution containing 15% to 40% (v / v) ammonium hydroxide. In a preferred embodiment, the basic agent solution contains 20% to 50% (v / v) ammonium hydroxide in water. In a more preferred embodiment, the basic agent solution contains about 25% (v / v) ammonium hydroxide in water.

[0075] In some preferred embodiments, a solution having a pH of about 11 or higher is obtained by contacting a plasma sample containing amyloid beta peptide with a basic denaturing agent. In more preferred embodiments, the pH of the solution may be about 11 to about 13. For example, the pH of the solution may be about 11 to about 12. The pH of the solution may be about 11 to 11.5, most preferably about 11.3.

[0076] The amount of basic denaturant used in the method of the present invention depends on the amount of plasma sample used and the pH of the basic denaturant. Those skilled in the art can determine the amount of basic denaturant to obtain a solution with a specific pH by simple calculation. In some preferred embodiments of the present invention, the basic denaturant is an aqueous solution of ammonium hydroxide at a concentration of 15% to 40% (v / v). In a more preferred embodiment of the present invention, a plasma sample in an amount of 100 μl to 400 μl is contacted with an aqueous solution of ammonium hydroxide at a concentration of 15% to 40% (v / v) in an amount of 200 μl to 800 μl.

[0077] The use of ammonium hydroxide as a denaturant to denature the plasma samples of the present invention provides several advantages, for example, many Aβ-plasma protein interactions (i.e., IgG) are disrupted (no precipitation occurs) while keeping the Aβ peptide in solution.

[0078] After denaturing the sample, the amyloid beta peptide is separated from other components of the sample by solid phase extraction (SPE).

[0079] Several types of SPE are known in the art. They can be classified according to the chemical properties of the stationary phase used. Thus, in normal-phase SPE, the stationary phase is more polar than the mobile phase, e.g., silica gel or alumina as the stationary phase, combined with a less polar mobile phase eluent (i.e., hexane). In contrast, reversed-phase SPE uses less polar packings, such as octadecylsilane or octylsilane bound to silica or polymer beads, and the mobile phase is typically a mixture of water and a miscible organic solvent and modifier. On the other hand, during ion-exchange SPE, components are separated based on electrostatic interactions between the components and positively or negatively charged groups on the stationary phase. Thus, ion-exchange SPE includes anion-exchange SPE, in which the stationary phase contains positively charged groups that interact with and retain negatively charged anions, such as acids, and cation-exchange SPE, in which the stationary phase contains negatively charged groups that interact with and retain positively charged cations, such as bases. Strong cation exchange sorbents contain aliphatic sulfonic acid groups that are always negatively charged in aqueous solution, while weak cation exchange sorbents contain aliphatic carboxylic acids that become charged at pH levels above 5. It is also possible to combine multiple retention mechanisms in the same cartridge, known as mixed-mode SPE, often combining reversed-phase and ion-exchange cartridges. Thus, mixed-mode reversed-phase anion exchange and mixed-mode reversed-phase cation exchange are known SPE methods in the art.

[0080] The purpose of solid phase extraction in the method of the present invention is to separate and discard undesirable components of the plasma sample in order to purify and enrich the plasma sample in the amyloid beta peptide of interest.

[0081] In some embodiments, the method of the present invention comprises a step of performing a first solid-phase extraction step on the solution obtained after the step of contacting the sample with a denaturing agent. In a preferred embodiment, the method of the present invention comprises a step of performing a second solid-phase extraction step after the first solid-phase extraction step. Thus, in some embodiments, the method of the present invention for preparing a sample containing amyloid beta peptide for analysis by mass spectrometry comprises two consecutive solid-phase extraction steps.

[0082] In some embodiments, the first and second solid phase extraction steps of the method of the present invention for preparing a sample containing amyloid beta peptides can be of any type known to those skilled in the art.

[0083] In some embodiments, the first solid phase extraction step is reversed-phase SPE. In some embodiments, the first solid phase extraction step is cation exchange SPE. In some embodiments, the first solid phase extraction step is anion exchange SPE. In some embodiments, the cation exchange SPE is strong cation exchange, weak cation exchange, or mixed-mode reversed-phase cation exchange. In some embodiments, the anion exchange SPE is strong anion exchange, weak anion exchange, or mixed-mode reversed-phase anion exchange.

[0084] In some embodiments, the second solid phase extraction step is reversed-phase SPE. In some embodiments, the second solid phase extraction step is cation exchange SPE. In some embodiments, the second solid phase extraction step is anion exchange SPE. In some embodiments, the cation exchange SPE is strong cation exchange, weak cation exchange, or mixed-mode reversed-phase cation exchange. In some embodiments, the anion exchange SPE is strong anion exchange, weak anion exchange, or mixed-mode reversed-phase anion exchange.

[0085] In some embodiments, the first solid phase extraction step is reversed-phase SPE and the second solid phase extraction step is cation exchange SPE, more preferably, the cation exchange SPE is strong cation exchange, weak cation exchange, or mixed-mode reversed-phase cation exchange.

[0086] In some embodiments, the first solid phase extraction step is reversed-phase SPE and the second solid phase extraction step is anion exchange SPE, more preferably the anion exchange SPE is strong anion exchange, weak anion exchange, or mixed mode reversed-phase anion exchange.

[0087] In some embodiments, the first solid phase extraction step is a cation exchange SPE, more preferably, the cation exchange SPE is a strong cation exchange, a weak cation exchange, or a mixed mode reversed phase-cation exchange, and the second solid phase extraction step is an anion exchange SPE, more preferably, the anion exchange SPE is a strong anion exchange, a weak anion exchange, or a mixed mode reversed phase-anion exchange.

[0088] In some embodiments, the first solid phase extraction step is anion exchange SPE, more preferably the anion exchange SPE is strong anion exchange, weak anion exchange, or mixed mode reversed phase-anion exchange, and the second solid phase extraction step is cation exchange SPE, more preferably the cation exchange SPE is strong cation exchange, weak cation exchange, or mixed mode reversed phase-cation exchange.

[0089] In some preferred embodiments of the methods of the present invention for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, the plasma sample is contacted with an acidic denaturant disclosed herein before performing the first and second SPEs, wherein the first SPE is reversed-phase SPE and the second solid-phase extraction step is cation exchange SPE.

[0090] In some preferred embodiments of the methods of the present invention for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, the plasma sample is contacted with an acidic denaturant disclosed herein before performing the first and second SPEs, wherein the first SPE is reversed-phase SPE and the second solid-phase extraction step is anion-exchange SPE.

[0091] In some preferred embodiments of the methods of the present invention for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, the plasma sample is contacted with an acidic denaturant disclosed herein before performing the first and second SPEs, wherein the first SPE is a cation exchange SPE and the second solid phase extraction step is an anion exchange SPE.

[0092] In some preferred embodiments of the methods of the present invention for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, the plasma sample is contacted with a basic denaturant disclosed herein before performing the first and second SPEs, wherein the first SPE is anion exchange SPE and the second solid phase extraction step is cation exchange SPE.

[0093] The specific combination of two consecutive SPE steps described herein, followed by denaturation of proteins in a plasma sample containing amyloid peptides according to the method of the present invention, allows for the purification of intact Aβ40 and Aβ42 peptides, which can be further analyzed by mass spectrometry without the need for additional purification steps.

[0094] The protocol for carrying out the first and second SPE steps of the present invention includes conditioning and equilibration of the stationary phase, loading the sample into the column or cartridge, at least one washing step, and at least one elution step, which are well known to those skilled in the art, and the specific conditions for each step are also known in the art.

[0095] In one embodiment of the present invention, protocols for carrying out the first and second SPE steps of the present invention are known in the art. In another embodiment of the present invention, the first and second SPE steps include at least one wash step to remove undesired components from the sample. In a preferred embodiment of the present invention, the first and second SPE steps each include at least two wash steps.

[0096] Suitable wash solutions are known in the art. In some embodiments, the wash solution for the SPE step of the method of the present invention is a solution of an acid, such as an organic acid, preferably acetic acid, formic acid, or trifluoroacetic acid (TFA). In other embodiments, the wash solution for the SPE step of the method of the present invention is a solution of water and a water-miscible organic solvent. For example, the water-miscible organic solvent can be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of classes of water-miscible organic solvents include, but are not limited to, esters, nitriles, carboxylic acids, amides, aldehydes, ketones, and combinations thereof. Those skilled in the art will understand that not all members of the above-listed classes are water-miscible, but those skilled in the art can easily determine which members of a particular class are water-miscible. In a more preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof. In a particularly preferred embodiment, the water-miscible organic solvent of the wash solution for the SPE step of the method of the present invention is acetonitrile. In other embodiments, the wash solution for the SPE step of the method of the present invention is a solution of a base, such as a water-soluble hydroxide, carbonate, oxide, and combinations thereof. In a preferred embodiment, the wash solution for the SPE step of the method of the present invention is an ammonium hydroxide solution.

[0097] In some preferred embodiments, the wash solution for the first wash is trifluoroacetic acid (TFA). The concentration of TFA that can be used for the first wash during the SPE step of the present invention is 0.01% to 10% (v / v) TFA in water. In another preferred embodiment, the wash solution is an aqueous solution of 0.05% to 1% (v / v) TFA. In a further preferred embodiment, the wash solution is an aqueous solution of about 0.1% (v / v) TFA. In a preferred embodiment, the wash solution for the first wash during the first SPE step of the present invention is an aqueous solution of 0.05% to 1% (v / v) TFA, more preferably about 0.1% TFA.

[0098] In some preferred embodiments, the wash solution for the first wash is formic acid. The concentration of formic acid usable for the first wash during the SPE step of the present invention is 15% to 35% (v / v) formic acid in water. In preferred embodiments, the wash solution is a 20% to 30% (v / v) aqueous solution of formic acid. In more preferred embodiments, the wash solution is about 25% (v / v) aqueous solution of formic acid. In another preferred embodiment, the wash solution for the first wash during the second SPE step of the present invention is a 20% to 30% (v / v) aqueous solution of formic acid, more preferably about 25% (v / v) aqueous solution of formic acid.

[0099] In some preferred embodiments, the wash solution for the first wash is an ammonium hydroxide solution. The concentration of ammonium hydroxide that can be used for the first wash during the SPE step of the present invention is 2% to 50% (v / v) ammonium hydroxide in water. In preferred embodiments, the wash solution is a 2% to 30% (v / v) aqueous solution of ammonium hydroxide. In more preferred embodiments, the wash solution is about 10% (v / v) aqueous solution of ammonium hydroxide. In another preferred embodiment, the wash solution for the first wash during the second SPE step of the present invention is a 5% to 30% (v / v) aqueous solution of ammonium hydroxide, more preferably about 10% (v / v) aqueous solution of ammonium hydroxide.

[0100] In some preferred embodiments, the wash solution for the second wash is acetonitrile. The concentration of acetonitrile that can be used for the second wash during the SPE step of the present invention is 5% to 80% (v / v) acetonitrile in water. In preferred embodiments, the wash solution is an aqueous solution of 10% to 60% (v / v) acetonitrile.

[0101] In a preferred embodiment, the washing solution for the second wash in the first SPE step of the present invention is an aqueous solution of 5-15% (v / v) acetonitrile. In a more preferred embodiment, the washing solution is an aqueous solution of about 10% (v / v) acetonitrile.

[0102] In a preferred embodiment, the washing solution for the second wash in the second SPE step of the present invention is an aqueous solution of 40% to 70% (v / v) acetonitrile. In a more preferred embodiment, the washing solution is an aqueous solution of about 60% (v / v) acetonitrile.

[0103] In another preferred embodiment, a third wash is performed during the first SPE step of the present invention. In a preferred embodiment, the wash solution for the third wash comprises acetonitrile at a concentration of 90% to 100% (v / v). In a more preferred embodiment, the wash solution for the third wash during the first SPE step is acetonitrile at a concentration of about 100% (v / v).

[0104] In a preferred embodiment, the first SPE step comprises at least two washing steps, the first washing step being performed with a solution containing 0.05% to 1% (v / v) TFA in water, the second washing step being performed with a solution containing 5 to 15% (v / v) acetonitrile in water, and the second SPE step comprises at least three washing steps, the first washing step being performed with a solution containing 20% ​​to 30% (v / v) formic acid in water, the second washing step being performed with a solution containing 40% to 70% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing 90% to 100% (v / v) acetonitrile.

[0105] In a more preferred embodiment, the first SPE step comprises at least two washing steps, the first with about 0.1% (v / v) TFA in water, the second with about 10% (v / v) acetonitrile in water, and the second SPE step comprises at least three washing steps, the first with about 25% (v / v) formic acid in water, the second with about 60% (v / v) acetonitrile in water, and the third with about 100% (v / v) acetonitrile.

[0106] In a preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing 0.05% to 10% (v / v) methanol in water, the second washing step being performed with a solution containing 5 to 15% (v / v) acetonitrile in water, and the second SPE step includes at least three washing steps, the first washing step being performed with a solution containing 0.5% to 50% (v / v) formic acid in water, the second washing step being performed with a solution containing 30% to 70% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing 90% to 100% (v / v) acetonitrile.

[0107] In a preferred embodiment, the first SPE step comprises at least three washing steps, the first with 5%-50% (v / v) formic acid in water, the second with 40-70% (v / v) acetonitrile in water, and the third with 90-100% (v / v) acetonitrile; the second SPE step comprises at least two washing steps, the first with 0.5%-15% (v / v) ammonium hydroxide in water, and the second with 30-70% (v / v) acetonitrile in water.

[0108] In a more preferred embodiment, the first SPE step comprises at least three washing steps, the first with about 25% (v / v) FA in water, the second with about 60% (v / v) acetonitrile in water, and the third with about 100% (v) acetonitrile; and the second SPE step comprises at least two washing steps, the first with about 10% (v / v) ammonium hydroxide in water, and the second with about 60% (v / v) acetonitrile in water.

[0109] In a more preferred embodiment, the first SPE step comprises at least two washing steps, the first washing step being performed with a solution containing 1% to 20% (v / v) ammonium hydroxide in water, the second washing step being performed with a solution containing 30 to 70% (v / v) acetonitrile in water, and the second SPE step comprises at least three washing steps, the first washing step being performed with a solution containing 0.5% to 50% (v / v) formic acid in water, the second washing step being performed with a solution containing 30% to 70% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing 90% to 100% (v / v) acetonitrile.

[0110] In a more preferred embodiment, the first SPE step comprises at least two washing steps, the first with about 10% (v / v) ammonium hydroxide in water, the second with about 60% (v / v) acetonitrile in water, and the second SPE step comprises at least three washing steps, the first with about 25% (v / v) formic acid in water, the second with about 60% (v / v) acetonitrile in water, and the third with about 100% (v / v) acetonitrile.

[0111] Any SPE step requires at least one washing step followed by elution of the analytes retained on the stationary phase. Suitable elution solutions are well known to those skilled in the art.

[0112] In some embodiments, the analytes retained on the stationary phase during the first SPE step are eluted with an elution solution comprising a surfactant and a water-miscible polar organic solvent. In other embodiments, the analytes retained on the stationary phase during the first SPE step are eluted with an elution solution comprising a base and a water-miscible polar organic solvent. In other embodiments, the analytes retained on the stationary phase during the first SPE step are eluted with an elution solution comprising an acid and a water-miscible polar organic solvent.

[0113] For example, the water-miscible organic solvent can be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of classes of water-miscible organic solvents include, but are not limited to, esters, nitriles, carboxylic acids, amides, aldehydes, ketones, and combinations thereof. Those skilled in the art will understand that not all members of the above-listed classes are water-miscible, but those skilled in the art can easily identify members of a particular class that are water-miscible. In a more preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof. In a particularly preferred embodiment, the water-miscible organic solvent of the elution solution after the first SPE step of the method of the present invention is acetonitrile. The surfactant of the elution solution can be of any type known to those skilled in the art, preferably a nonionic surfactant. In some embodiments, the surfactant is nonionic. In some preferred embodiments, the nonionic surfactant is an ethoxylate type. In a more preferred embodiment, the ethoxylate type nonionic surfactant is Triton X-100. The base can be selected from the group consisting of water-soluble hydroxides, carbonates, oxides, and combinations thereof. For example, the base can be a water-soluble hydroxide. Suitable hydroxides include inorganic hydroxides, organic hydroxides, and combinations thereof. In a preferred embodiment, the base is ammonium hydroxide. The acid of the elution solution can be an organic acid, preferably acetic acid, formic acid, or trifluoroacetic acid (TFA).

[0114] In a preferred embodiment, the elution solution used after the first SPE step of the method of the present invention comprises Triton X-100 at a concentration of 1% to 5% (v / v) and acetonitrile at a concentration of 20% to 40% (w / v). In a more preferred embodiment, the elution solution consists of an aqueous solution of about 2% (v / v) Triton X-100 and about 30% (w / v) acetonitrile.

[0115] In another preferred embodiment, the elution solution used after the first SPE step of the method of the present invention comprises ammonium hydroxide at a concentration of 5% to 15% (v / v) and acetonitrile at a concentration of 50% to 90% (w / v). In a more preferred embodiment, the elution solution consists of an aqueous solution of about 10% (v / v) ammonium hydroxide and about 75% (w / v) acetonitrile.

[0116] In another preferred embodiment, the elution solution used after the first SPE step of the method of the present invention comprises a trifluoroacetic acid concentration of 1% to 15% (v / v) and an acetonitrile concentration of 50% to 90% (w / v). In a more preferred embodiment, the elution solution consists of an aqueous solution of about 5% (v / v) trifluoroacetic acid and about 70% (w / v) acetonitrile.

[0117] In some embodiments, the analytes retained on the stationary phase during the second SPE step are eluted with an elution solution comprising a base and a water-miscible polar organic solvent, while in other embodiments, the analytes retained on the stationary phase during the second SPE step are eluted with an elution solution comprising an acid and a water-miscible polar organic solvent.

[0118] The base may be selected from the group consisting of water-soluble hydroxides, carbonates, oxides, and combinations thereof. For example, the base may be a water-soluble hydroxide. Suitable hydroxides include inorganic hydroxides, organic hydroxides, and combinations thereof. In a preferred embodiment, the base is ammonium hydroxide. The water-miscible organic solvent may be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of classes of water-miscible organic solvents include, but are not limited to, esters, nitriles, carboxylic acids, amides, aldehydes, ketones, and combinations thereof. Those skilled in the art will understand that not all members of the classes set forth above are water-miscible, but those skilled in the art can easily identify members of a particular class that are water-miscible. In a more preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof. In a particularly preferred embodiment, the water-miscible organic solvent of the elution solution after the second SPE step of the method of the present invention is acetonitrile. The acid of the elution solution may be an organic acid, preferably acetic acid, formic acid, or trifluoroacetic acid (TFA).

[0119] In a preferred embodiment, the elution solution used after the second SPE step of the method of the present invention comprises ammonium hydroxide at a concentration of 5% to 15% (v / v) and acetonitrile at a concentration of 50% to 90% (w / v). In a more preferred embodiment, the elution solution consists of an aqueous solution of about 10% (v / v) ammonium hydroxide and about 75% (w / v) acetonitrile.

[0120] In another preferred embodiment, the elution solution used after the first SPE step of the method of the present invention comprises a trifluoroacetic acid concentration of 1% to 15% (v / v) and an acetonitrile concentration of 50% to 90% (w / v). In a more preferred embodiment, the elution solution consists of an aqueous solution of about 5% (v / v) trifluoroacetic acid and about 70% (w / v) acetonitrile.

[0121] After elution of the analytes of interest during the first SPE step of the present invention, the pH of the resulting eluate can be adjusted to prepare the eluate for subsequent steps. In a preferred embodiment, the pH of the solution containing the analytes of interest is reduced by contacting the eluate obtained after elution during the first SPE with an aqueous solution of formic acid, preferably an aqueous solution of formic acid at a concentration of 30% to 70% (v / v), more preferably an aqueous solution of formic acid at a concentration of about 50% (v / v).

[0122] The eluate containing purified intact amyloid beta peptide obtained after the second SPE step can then be dried and resuspended in a solution suitable for downstream analysis, such as mass spectrometry. Suitable drying methods are known in the art and include, but are not limited to, evaporation in a centrifugal vacuum concentrator (e.g., Thermo SpeedVac, Genevac) and lyophilization. In a preferred embodiment, the eluate obtained after the second SPE step of the method of the present invention for preparing a sample containing amyloid beta peptide is dried in a vacuum concentrator for at least 30 minutes at a temperature of 30°C to 50°C. In a more preferred embodiment, the eluate is dried in a vacuum concentrator for approximately 35 minutes at a temperature of 40°C to 47°C.

[0123] After drying the eluate, it is necessary to resuspend the analyte in a solution suitable for the particular downstream analysis. In some embodiments, the downstream analysis can be an antibody-based detection method such as ELISA, but in more preferred embodiments, the downstream analysis is mass spectrometry, including a combination of liquid chromatography and mass spectrometry. Suitable solutions for different downstream analyses are known in the art.

[0124] In some embodiments of the invention, the analyte is further subjected to mass spectrometry, including a combination of liquid chromatography and mass spectrometry, and the solution in which the dried analyte is resuspended comprises at least two of a surfactant, a reducing agent, a water-miscible polar organic solvent, and an acid. In other embodiments, the solution in which the dried analyte is resuspended comprises a surfactant and a reducing agent. In other embodiments, the solution in which the dried analyte is resuspended comprises a surfactant, a reducing agent, a polar organic solvent, and an acid.

[0125] According to the present invention, the surfactant of the solution in which the dried analyte is resuspended can be of any type known to those skilled in the art, preferably non-ionic. In some embodiments, the surfactant is non-ionic. In some preferred embodiments, the non-ionic surfactant is of the ethoxylate type. In a further preferred embodiment, the ethoxylate type non-ionic surfactant is Triton X-100.

[0126] According to the present invention, the reducing agent of the solution in which the dried analyte is resuspended can be of any type known to those skilled in the art, preferably a reducing agent suitable for reducing disulfide bonds. Non-limiting examples of reducing agents suitable for reducing disulfide bonds include organophosphine reducing agents or other reducing agents, such as dithiothreitol or β-mercaptoethanol. In a preferred embodiment, the reducing agent suitable for reducing disulfide bonds is an organophosphine reducing agent. In a more preferred embodiment, the organophosphine reducing agent is tris-carboxyethylphosphine.

[0127] According to the present invention, the water-miscible polar organic solvent of the solution in which the dried analyte is resuspended can be any type known to those skilled in the art, preferably acetonitrile or dimethylformamide. In other embodiments, the polar organic solvent can be a mixture of acetonitrile and dimethylformamide. The water-miscible organic solvent can be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of classes of water-miscible organic solvents include, but are not limited to, ethers, esters, nitriles, carboxylic acids, amides, aldehydes, ketones, and combinations thereof. Those skilled in the art will understand that not all members of the above-listed classes are water-miscible, but those skilled in the art can easily determine which members of a particular class are water-miscible. In a further preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof.

[0128] According to the present invention, the acid of the solution in which the dried analyte is resuspended can be any type known to those skilled in the art, preferably an organic acid. In a more preferred embodiment, the organic acid is a carboxylic acid. In a more preferred embodiment, the carboxylic acid is a monocarboxylic acid, a dicarboxylic acid, or a tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid can have a pKa of about 4 or less, for example, about 3 or less, for example, about 2 or less, and preferably 1 or less. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid can have a pKa of about 0.5 or less. In a preferred embodiment, the carboxylic acid is a C1-C10 haloalkyl monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. For example, the carboxylic acid can be a C1-C105 haloalkyl monocarboxylic acid. In a more preferred embodiment, the carboxylic acid is trifluoroacetic acid (TFA). Thus, in some preferred embodiments, the acid of the solution in which the dried analyte is resuspended is TFA.

[0129] All pKa values ​​disclosed herein are measured in water at 25° C. and 1 atm pressure.

[0130] In a more preferred embodiment, the surfactant is Triton X-100, the reducing agent is tris-carboxyethylphosphine, the polar organic solvents are acetonitrile and dimethylformamide, and the acid is TFA.

[0131] In some preferred embodiments, the concentration of acetonitrile in the solution in which the dried analyte is dissolved is between 2% and 8% (v / v), more preferably between 3% and 7% (v / v), more preferably between 4% and 6% (v / v), and even more preferably about 5% (v / v).

[0132] In some preferred embodiments, the concentration of dimethylformamide in the solution in which the dried analyte is dissolved is between 0.1% and 3% (v / v), more preferably between 0.5% and 2% (v / v), more preferably between 0.5% and 1.5% (v / v), and even more preferably about 1% (v / v).

[0133] In some preferred embodiments, the concentration of trifluoroacetic acid (TFA) in the solution in which the dried analyte is dissolved is between 0.1% and 5% (v / v), more preferably between 0.2% and 4% (v / v), more preferably between 0.2% and 3% (v / v), more preferably between 0.2% and 2.5% (v / v), and even more preferably about 0.5% (v / v).

[0134] In some preferred embodiments, the concentration of Triton X-100 in the solution in which the dried analyte is dissolved is between 0.01% and 2% (v / v), more preferably between 0.05% and 1% (v / v), more preferably between 0.05% and 0.8% (v / v), more preferably between 0.05% and 0.1% (v / v), and even more preferably about 0.05% (v / v).

[0135] In some preferred embodiments, the concentration of tris-carboxyethylphosphine in the solution in which the dried analyte is dissolved is between 0.05% and 0.3% (w / v), more preferably between 0.1% and 0.2% (w / v), more preferably between 0.12% and 0.16% (w / v), and even more preferably about 0.14% (w / v).

[0136] In some preferred embodiments, the solution in which the analyte is dissolved is an aqueous solution containing a surfactant and a reducing agent. In further preferred embodiments, the solution in which the dried analyte is dissolved contains Triton X-100 at a concentration of 0.05% to 0.8% (v / v) and tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v).

[0137] In another preferred embodiment, the solution in which the dried analyte is dissolved contains Triton X-100 at a concentration of 0.01% to 0.1% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), and acetonitrile at a concentration of 3% to 7% (v / v).

[0138] In some preferred embodiments, the solution in which the analyte is dissolved is an aqueous solution containing a surfactant, a reducing agent, a polar organic solvent, and an acid. In more preferred embodiments, the solution in which the dried analyte is dissolved contains Triton X-100 at a concentration of 0.01% to 0.1% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v). In a further preferred embodiment, the solution in which the dried analyte is dissolved comprises Triton X-100 at a concentration of 0.01% to 0.1% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), dimethylformamide at a concentration of 0.1% to 3% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v).

[0139] In some embodiments, samples prepared by the methods of the present invention are further analyzed by chromatography and / or mass spectrometry.

[0140] The present invention also relates to a method for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, characterized in that the method does not involve immunoprecipitation or digestion of the plasma sample prior to analysis by mass spectrometry.

[0141] The present invention also provides a method for preparing a plasma sample containing amyloid beta peptide for analysis by mass spectrometry, comprising the steps of: a) contacting the plasma sample with a denaturing agent; b) subjecting the solution obtained in step a) to a first solid phase extraction step to recover a first eluate; c) subjecting the first eluate obtained in step b) to a second solid phase extraction step to recover a second eluate; and d) drying the second eluate obtained in step c) and processing it for analysis by mass spectrometry; The method is characterized in that the sample obtained from step d) contains intact amyloid beta peptides Aβ40 and Aβ42.

[0142] In a second aspect, the present invention relates to a method for the mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample. In some embodiments, the method of the present invention for the mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample comprises steps a) to d) of the method described herein for preparing the sample, and further comprises: i) subjecting the solution obtained in step d) to a liquid chromatography step to separate the analytes of interest; ii) subjecting the analytes separated in step i) to ionization to generate one or more charged species; iii) separating the one or more charged species according to ion mobility; iv) detecting the one or more charged species separated in step iii) and determining their abundance by mass spectrometry; and v) determining the amount or concentration of intact amyloid beta peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged species measured in step iv) with a standard curve.

[0143] In some embodiments of the present invention, the method for mass spectrometric quantification of intact amyloid peptides Aβ40 and Aβ42 in a plasma sample comprises subjecting a solution resulting from the method described herein for preparing a sample comprising amyloid beta peptides to a chromatography step i). In a preferred embodiment, the chromatography step is liquid chromatography (LC). In a more preferred embodiment, the liquid chromatography is HPLC. In an even more preferred embodiment, the chromatography step is micro liquid chromatography (micro HPLC).

[0144] In some embodiments of the present invention, the method for mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in plasma samples comprises a second step ii) of subjecting the analytes separated in step i) to ionization to generate one or more charged species.

[0145] In some embodiments of the present invention, the ionization to which the analyte is subjected is electrospray ionization (ESI) in positive ion mode. Conditions for carrying out electrospray ionization according to the present invention are well known to those skilled in the art.

[0146] In some embodiments of the present invention, the method for mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample comprises a third step iii) of separating one or more charged species obtained in step ii) according to ion mobility. In a preferred embodiment, the one or more charged species are separated by ion mobility spectrometry (IMS). In a more preferred embodiment, the ion mobility spectrometry technique used in the method of the present invention is differential mobility spectrometry (DMS).

[0147] In some embodiments of the invention, after separating the charged species according to their ion mobility, they are detected and their abundances are determined by mass spectrometry (step iv). As previously explained, the term "mass spectrometry" encompasses several analytical techniques for determining the mass-to-charge ratio of an analyte or group of analytes. Non-limiting examples of mass spectrometry techniques include ion traps (3D or linear), single or triple quadrupoles, time-of-flight, Orbitrap, Fourier transform ion cyclotron resonance mass spectrometry, and combinations thereof (hybrid instruments).

[0148] In a preferred embodiment, the technique for measuring the intensities and abundances of charged species by mass spectrometry is multiple reaction monitoring (MRM) in a triple quadrupole instrument.

[0149] In some embodiments of the present invention, the method for mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in plasma samples comprises a fifth step v) in which the amount or concentration of the peptides is determined by comparison of the analyte abundance measured in iv) with a standard curve.

[0150] Therefore, in order to quantify the amount or concentration of an analyte in a sample analyzed by mass spectrometry, it is preferable to generate a calibration curve using at least one standard substance with increasing concentrations. In the present invention, Aβ40 and Aβ42 are peptides that are preferably quantified, and therefore, labeled Aβ40 and Aβ42 are preferably quantified. 15 N-Aβ40 and 15 N-Aβ42 is preferably used as the standard for the calibration curve.

[0151] As previously described in the prior art, a calibration curve can be generated using a buffer solution such as PBS containing BSA. However, in a more preferred embodiment of the present invention, generating a calibration curve using plasma offers several advantages, such as equal recovery of both the standard and the analyte, and equalizing matrix effects that may adversely affect quantification. In a more preferred embodiment, the calibration curve is generated using human plasma.

[0152] Furthermore, internal standards are used as controls for the quality of the various steps of the method of the invention and for signal normalization. Thus, internal standards can be added to solutions to generate calibration curves and to samples to be analyzed by mass spectrometry to ensure the quality of the various steps.

[0153] In some embodiments, the calibration curve solutions and samples are labeled. 2 H-Aβ40 and 2 H-Aβ42 is added as an internal standard. In another embodiment, the calibration curve solution and the sample are labeled. 13 C-Aβ40 and 13 C-Aβ42 is added as an internal standard.2 H-Aβ40 and 13 C-Aβ42, or 2 H-Aβ42 and 13 C-Aβ40 is added as an internal standard.

[0154] In another embodiment, the present invention relates to a method for the mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample, characterized in that the method does not comprise a step of determining the amount or concentration of the peptides by comparison of the analyte abundance measured in iv) with a standard curve. In this embodiment, the method for quantification is a semi-quantitative method in which the use of an internal standard consists in quantifying the analyte abundance without comparison with a standard curve.

[0155] The sample containing amyloid beta peptide used in the method of the present invention is preferably a plasma sample. The amount of plasma sample used in step a) of the method for preparing a sample for analysis by mass spectrometry can be determined by a person skilled in the art taking into account the specific protocol of the mass spectrometry to be followed.

[0156] However, one advantage of the method of the present invention is that it can reduce the amount of sample required to obtain accurate values ​​for the concentrations of amyloid beta peptides Aβ40 and Aβ42 in order to identify early stages of neurodegenerative diseases such as Alzheimer's disease in a subject. The reduced amount of plasma sample required in the method of the present invention is particularly advantageous for large-scale screening studies.

[0157] Thus, the sample volume used in step a) of the method for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry is between 100 μL and 400 μL. In a preferred embodiment, the sample volume is between 150 μL and 300 μL. In a more preferred embodiment, the sample volume is between 200 μL and 250 μL. In an even more preferred embodiment, the sample volume is about 200 μL.

[0158] In a third aspect, the present invention relates to an aqueous solution for treating a dried eluate to be analyzed by mass spectrometry, the aqueous solution comprising at least two of a surfactant, a reducing agent, a water-miscible polar organic solvent, and an acid. In another embodiment, the aqueous solution comprises a surfactant and a reducing agent. In another embodiment, the solution comprises a surfactant, a reducing agent, a polar organic solvent, and an acid.

[0159] According to the present invention, the surfactant of the solution in which the dried analyte is processed can be of any type known to those skilled in the art, preferably non-ionic. In some embodiments, the surfactant is non-ionic. In some preferred embodiments, the non-ionic surfactant is of the ethoxylate type. In a further preferred embodiment, the ethoxylate type non-ionic surfactant is Triton X-100.

[0160] According to the present invention, the reducing agent of the solution in which the dried analyte is treated can be of any type known to those skilled in the art, preferably a reducing agent suitable for reducing disulfide bonds. Non-limiting examples of reducing agents suitable for reducing disulfide bonds include organophosphine reducing agents or other reducing agents, such as dithiothreitol or β-mercaptoethanol. In a preferred embodiment, the reducing agent suitable for reducing disulfide bonds is an organophosphine reducing agent. In a more preferred embodiment, the organophosphine reducing agent is tris-carboxyethylphosphine.

[0161] According to the present invention, the water-miscible polar organic solvent of the solution in which the dried analyte is processed can be of any type known to those skilled in the art. The water-miscible organic solvent can be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of classes of water-miscible organic solvents include, but are not limited to, esters, nitriles, carboxylic acids, amides, aldehydes, ketones, and combinations thereof. Those skilled in the art will understand that not all members of the classes set forth above are water-miscible, but those skilled in the art can easily determine which members of a particular class are water-miscible. In a more preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof.

[0162] According to the present invention, the acid of the solution in which the dried analyte is treated can be any type known to those skilled in the art, preferably an organic acid. In a more preferred embodiment, the organic acid is a carboxylic acid. In a more preferred embodiment, the carboxylic acid is a monocarboxylic acid, a dicarboxylic acid, or a tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid can have a pKa of about 4 or less, for example, about 3 or less, for example, about 2 or less, and preferably 1 or less. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid can have a pKa of about 0.5 or less. In a preferred embodiment, the carboxylic acid is a C1-C10 haloalkyl monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. For example, the carboxylic acid can be a C1-C5 haloalkyl monocarboxylic acid. In a more preferred embodiment, the carboxylic acid is trifluoroacetic acid (TFA). Thus, in some preferred embodiments, the acid of the solution in which the dried analyte is treated is TFA.

[0163] In a more preferred embodiment, the surfactant is Triton X-100, the reducing agent is tris-carboxyethylphosphine, the polar organic solvents are acetonitrile and dimethylformamide, and the acid is TFA.

[0164] In a more preferred embodiment, the aqueous solution of the invention for resuspending the dried eluate to be analyzed by mass spectrometry comprises Triton X-100 at a concentration of 0.01% to 0.1% (v / v) and tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v).

[0165] In another preferred embodiment, the aqueous solution of the present invention for treating the dried eluate to be analyzed by mass spectrometry comprises Triton X-100 at a concentration of 0.010% to 1% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), dimethylformamide at a concentration of 0.1% to 3% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v).

[0166] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. [Example]

[0167] Example 1 Preparation of plasma samples for analysis by mass spectrometry Calibration curves and quality control Human plasma and 15 N-Aβ40 and 15 N-Aβ42 labeled standards (rPeptide, Watkinsville, GA, USA) were used to prepare calibration curves and quality control samples. 15 N-Aβ40 and 15 Calibration ranges of 50-3000 pg / ml and 10-100 pg / ml were used to prepare N-Aβ42, respectively.

[0168] A series of standards for the calibration curve, quality control samples, and plasma samples from human subjects to be further analyzed were all custom-labeled as internal standards for quality control and signal normalization. 2 H-Aβ40 and 2 H-Aβ42 (Bachem, Bubendorf, Switzerland) was added.

[0169] Preparation of plasma samples for analysis by mass spectrometry Plasma samples obtained from human subjects were prepared according to different protocols (A, B, C or D) depending on the combination of SPE cartridges subsequently used.

[0170] Protocol A: First, the selected internal standard was added to the sample. Then, the sample was denatured by contacting 400 μl of 50% aqueous formic acid (FA) with 200 μl of human plasma. After denaturation, amyloid peptides were purified using two consecutive SPE steps.

[0171] The first SPE consisted of reversed-phase SPE using HLB Prime extraction plates (OASIS HLB Prime 96-well plates, 30 mg, Waters, Milford, MA, USA, part number 186008054). Suitable conditions for the first SPE are outlined below. Solvation: 1 ml of acetonitrile Adjustment: 1 ml of 0.1% FA (formic acid) solution Adding sample Wash 1: 0.5 ml of 5% methanol Wash 2: 0.5 ml of 10% acetonitrile solution Elution: 0.4 ml of 0.1% TFA-acetonitrile / water 70 / 30 solution Acidify the eluate by adding 10 μl of 50% FA solution.

[0172] After the first SPE, the eluate was subjected to a second SPE, in this case a mixed-mode reversed-phase-cation exchange SPE using an MCX extraction plate (OASIS MCX 96-well microelution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below. Solvation: 0.2 ml of methanol ·Adjustment: 5% FA aqueous solution 0.2ml Sample loading: Acidified eluate from SPE 1 Wash 1: 0.4 ml of 5% FA solution Wash 2: 0.4 ml of 40% acetonitrile solution Wash 3: 0.4 ml of 100% acetonitrile Elution: acetonitrile / water / ammonium hydroxide 75 / 15 / 10 100 μl

[0173] After the second SPE, the eluate was evaporated to dryness in a vacuum concentrator for 35 minutes at 45° C. The dried sample was then resuspended in 25 μl of AB solvent, an in-house optimized solution consisting of: 100% acetonitrile: 2.5ml Dimethylformamide (DMF) 100%: 0.5 ml ·TFA 25% aqueous solution: 1ml ·Triton X-100 10% aqueous solution: 0.25ml Tris-carboxyethylphosphine: 70mg Water: Make up to 50ml in a volumetric flask

[0174] Protocol B: First, the selected internal standard was added to the sample. Then, the sample was denatured by contacting 400 μl of 50% aqueous formic acid (FA) with 200 μl of human plasma. After denaturation, amyloid peptides were purified using two consecutive SPE steps.

[0175] The first SPE consisted of reversed-phase SPE using HLB extraction plates (OASIS HLB, 96-well plates, 30 μm, 30 mg, Waters, Milford, MA, USA, part number WAT058951). Suitable conditions for the first SPE are outlined below. Solvation: 1 ml of acetonitrile Adjustment: 1 ml of 0.1% TFA (trifluoroacetic acid) solution Adding sample Wash 1: 0.1% TFA 1ml Wash 2: 0.5 ml of 10% acetonitrile solution Elution: 0.7 ml of 2% Triton X-100-acetonitrile / water 30 / 70 solution Acidify the eluate by adding 10 μl of 50% FA solution.

[0176] After the first SPE, the eluate was subjected to a second SPE, in this case a mixed-mode reversed-phase-cation exchange SPE using an MCX extraction plate (OASIS MCX 96-well microelution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below. Solvation: 0.2 ml of methanol ·Adjustment: 25% FA aqueous solution 0.2ml Sample loading: Acidified eluate from SPE 1 Wash 1: 0.4 ml of 25% FA solution Wash 2: 0.4 ml of 60% acetonitrile solution Wash 3: 0.4 ml of 100% acetonitrile Elution: acetonitrile / water / ammonium hydroxide 75 / 15 / 10 100 μl

[0177] After the second SPE, the eluate was evaporated to dryness in a vacuum concentrator for 35 min at 45° C. The dried sample was then resuspended in 25 μl of AB solvent, an in-house optimized solution consisting of 5% v / v AcN, 1% v / v dimethylformamide, 0.5% v / v TFA, 0.05% v / v Triton X-100, and 0.14% w / v tris-carboxyethylphosphine in water.

[0178] Protocol C: First, the selected internal standard was added to the sample. Then, the sample was denatured by contacting 400 μl of 50% aqueous formic acid (FA) with 200 μl of human plasma. After denaturation, amyloid peptides were purified using two consecutive SPE steps.

[0179] The first SPE consisted of a mixed-mode reversed-phase-cation exchange SPE using MCX extraction plates (OASIS MCX 96-well plates, 30 μm, 30 mg, Waters, Milford, MA, USA, part number 186000248). Suitable conditions for the first SPE are outlined below. Solvation: 1 ml of methanol ·Adjustment: 1ml of 25% FA aqueous solution Adding sample Wash 1: 1 ml of 25% FA solution Wash 2: 1 ml of 60% acetonitrile water solution (60 / 40) Wash 3: 1 ml of 100% acetonitrile Elution: acetonitrile / water / ammonium hydroxide (75 / 15 / 10) 2x400μl

[0180] After the first SPE, the eluate was subjected to a second SPE, in this case a mixed-mode reversed-phase-anion exchange SPE using a MAX microelution plate (OASIS MAX Microelution Plate, Waters, Milford, Massachusetts, USA, part number 186001829). Suitable conditions for the second SPE are outlined below. Solvation: 0.3 ml of methanol ·Adjustment: NH4OH 10% 0.4ml Adding sample Wash 1: NH4OH 10% 0.4ml Wash 2: 0.4 ml of acetonitrile / water (60 / 40) Elution: 2x acetonitrile / water 70 / 30 50 μl, 5% TFA

[0181] After the second SPE, the eluate was evaporated to dryness in a vacuum concentrator for 35 min at 45° C. The dried sample was then resuspended in 25 μl of AB solvent, an in-house optimized solution consisting of 5% v / v AcN, 1% v / v dimethylformamide, 0.5% v / v TFA, 0.05% v / v Triton X-100, and 0.14% w / v tris-carboxyethylphosphine in water.

[0182] Protocol D: First, the selected internal standard was added to the sample. Then, the sample was denatured by contacting 400 μl of 25% aqueous NH4OH with 200 μl of human plasma. After denaturation, amyloid peptides were purified using two consecutive SPE steps.

[0183] The first SPE consisted of a mixed-mode reversed-phase-anion exchange SPE using OASIS MAX 96-well plates (30 μm, 30 mg, Waters, Milford, MA, USA, part number 186000373). Suitable conditions for the first SPE are outlined below. Solvation: 1 ml of methanol ·Adjustment: NH4OH 10% 1ml Adding sample Wash 1: NH4OH 10% 1ml Wash 2: 1 ml of acetonitrile / water 60 / 40 Elution: 2x acetonitrile / water 70 / 30 400 μl, 5% trifluoroacetic acid

[0184] After the first SPE, the eluate was subjected to a second SPE, in this case a mixed-mode reversed-phase-cation exchange SPE using an MCX extraction plate (OASIS MCX 96-well microelution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below. Solvation: 0.2 ml of methanol ·Adjustment: 25% FA aqueous solution 0.2ml Adding sample Wash 1: 0.4 ml of 25% FA solution Wash 2: 0.4 ml of 60% acetonitrile solution Wash 3: 0.4 ml of 100% acetonitrile Elution: acetonitrile / water / ammonium hydroxide 75 / 15 / 10 100 μl

[0185] After the second SPE, the eluate was evaporated to dryness in a vacuum concentrator for 35 minutes at 45° C. The dried sample was then resuspended in 25 μl of AB solvent, an in-house optimized solution consisting of: 100% acetonitrile: 2.5ml Dimethylformamide (DMF) 100%: 0.5 ml ·TFA 25% aqueous solution: 1ml ·Triton X-100 10% aqueous solution: 0.25ml Tris-carboxyethylphosphine: 70mg Water: Make up to 50ml in a volumetric flask

[0186] In both cases, the calibration curve and quality control samples were also subjected to the same preparation protocol.

[0187] Example 2 Analysis of plasma samples by mass spectrometry The plasma samples prepared in Example 1 were then analyzed by mass spectrometry according to the method of the present invention for the quantification of amyloid peptides.

[0188] The analytical system used for quantification of amyloid peptides consisted of the following modules: M3 micro HPLC dual-pump (trap-elution) chromatograph equipped with a CTC autosampler and column oven (Sciex, Framingham, MA, USA) 6500+ QTRAP Hybrid Triple Quadrupole-Linear Ion Trap Mass Spectrometer Coupled with SelexION Mobility Differential Spectroscopy-DMS Interface (Sciex, Framingham, MA, USA)

[0189] After injection, the sample was loaded onto a trap column (YMC Triart C18, 12 nm, 3 μm, 5 × 0.3 mm, YMC, Dinslaken, Germany). A trapping flow rate of 50 μl / min with an aqueous solution of 0.5% TFA and 5% dimethyl sulfoxide (DMSO) was used for 2 min. The trapping valve was then switched, and the analytes were eluted from the trap column to the analytical column and MS system. The following chromatographic conditions were used: ○A phase: 0.1% FA in water Phase B: 0.1% FA in acetonitrile ○Flow rate: 15μl / min ○Column: HALO Protein C18, 400Å, 3.4μm, 0.3x50mm (Advanced Materials Technology, Wilmington, DE, USA) Column temperature: 55°C Gradient: 15% B for 0.3 min, linear gradient to 40% B at 3.5 min, ramp to 90% B in 0.1 min, hold for 0.3 min, return to initial conditions (15% B) in 0.1 min, hold for 3 min to re-equilibrate the column. At 5.5 min, the analytical valve was switched on and the system was washed with 0.1% Triton X-100 in 80 / 20 TFE / water, after which the trap column was equilibrated with the additive solvent (5% DMSO, 0.5% TFA in water).

[0190] Sample acquisition began when the trapping valve was switched, allowing the analytes to elute onto the analytical column. After chromatographic separation, the analytes entered the ion source of the mass spectrometer. They were subjected to electrospray ionization (ESI) in positive ion mode. While in the gas phase, the analytes were subjected to differential mobility spectrometry (DMS) to ensure maximum separation from interfering matrix ions.

[0191] The specific combination of DMS and micro-LC reduced background noise and increased sensitivity.

[0192] After DMS separation, the ions (charged species of the analytes of interest) were analyzed by multiple reaction monitoring (MRM) in a triple quadrupole instrument. Briefly, precursor (pseudo-molecular) ions were filtered in the first quadrupole (Q1), fragmented in the second quadrupole (Q2 or collision cell) by collision with the target gas (a process known as collision-induced dissociation (CID) or collision-activated dissociation (CAD)), and the fragments of interest were filtered in the third quadrupole (Q3) and allowed to reach the detector.

[0193] Precursor ions with charge state 5 (z=5) were selected in Q1, fragmented by collision with nitrogen gas in Q2, and the fragments analyzed in Q3 (also with z=5). The MRM acquisition parameters are outlined below.

[0194] [Table 1]

[0195] Aβ40, 15 N-Aβ40 and 2 In H-Aβ40, b 39 5+ Product ions were analyzed in Q3. Aβ42, 15 N-Aβ42 and 2 In H-Aβ42, b 41 5+ Product ions were analyzed in Q3. All molecular masses in the table above are average masses.

[0196] The following acquisition parameters were the same for all species: source temperature 250°C, curtain gas 30 pounds per square inch (psi), ion spray voltage 4800 volts (v), ion source gas 1 (nebulization) 30 psi, ion source gas 2 (desolvation) 50 psi, declustering potential 85 v, and entrance potential 10 v. Both filtering quadrupoles Q1 and Q3 were operated at unit derivation.

[0197] The linear equation was calculated by linear regression of the calibration curve samples as the peak area ratio ( 15 N-Aβ40 / 2 H-Aβ40 and 15 N-Aβ42 / 2 H-Aβ42) versus concentration data. For quality control samples, the response ratio ( 15 N-Aβ40 / 2 H-Aβ40 and 15 N-Aβ42 / 2 H-Aβ42) was inserted into the corresponding calibration curve to obtain back-calculated concentrations and evaluated. 2 H-Aβ40 and Aβ42 / 2 H-Aβ42) was interpolated in the corresponding calibration curve to obtain the calculated concentration. Regression analysis was performed using MultiQuant 3.0.3 software (Sciex, Framingham, MA, USA).

[0198] Example 3 Quantification of intact amyloid beta peptides Aβ40 and Aβ42 in plasma samples prepared and analyzed according to the present invention Twenty replicates from two different plasma samples (sample A and sample B) were prepared according to the methods described in Example 1 (protocol B) and Example 2 and further analyzed by mass spectrometry according to the method described in Example 2.

[0199] The values ​​obtained by interpolation in the calibration curve of the quantification of amyloid beta peptides carried out on 20 replicates of each plasma sample are shown in Table 1 below.

[0200] As can be seen in Table 1, the coefficient of variation (%CV) for the quantification of Aβ40 is less than 4%, while the coefficient of variation for the quantification of Aβ42 is approximately 6%. These coefficients of variation represent low variability, making the method of the present invention suitable for detecting small differences in Aβ peptides between groups.

[0201] [Table 2]

[0202] The absolute quantification of Aβ40 in human plasma was similar to that obtained by MS and immunological ligand binding assay (LBA) methods (mostly ELISA) known in the art, whereas the quantification of Aβ42 was significantly higher than that observed by the method of the present invention.

[0203] Furthermore, while the method of the present invention allows for the quantification of intact peptides Aβ40 and Aβ42, other methods known in the art, such as the method of Bateman's group, detect truncated peptides (Ovod et al., Amyloid B concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis, Alzheimer's and Dementia, October 2017; 13(10): 1185).

[0204] Example 4 Detecting small changes in Aβ species in a sample Further studies investigated the ability to detect small differences in amyloid concentrations in human plasma. Therefore, 200 μl and 220 μl plasma samples were prepared according to the method in Example 1, Protocol B, and further analyzed by mass spectrometry according to the method described in Example 2. In this case, the analyte ratio ( 14 N-Aβ40 / 15 N-Aβ40 and 14 N-Aβ42 / 15 The area of ​​N-Aβ42 was compared.

[0205] In this example, the amyloid concentrations in the plasma samples were identical, but this was not true in absolute terms due to the 10% difference in the dosage used, which mimics the 10% concentration difference in real samples.

[0206] Table 2 shows the results of the quantification of Aβ42 and Aβ40 performed in several replicates of the same plasma sample, with starting sample volumes of 200 μl and 220 μl, according to the method of the invention described in Examples 1 and 2.

[0207] The results show that a 10% increase in the amount of plasma sample results in a 10% and 9% increase in the amount of Aβ42 and Aβ40, respectively, as quantified by the method of the present invention.

[0208] [Table 3]

[0209] Example 5 Diagnostic performance of the method of the present invention Samples from 36 individuals (26 PET-negative individuals and 14 PET-positive individuals) previously characterized by positron emission tomography (PET) were prepared according to the method described in Example 1 and further analyzed by mass spectrometry according to the method described in Example 2. The Aβ42 / Aβ40 ratio was calculated and is shown in Figure 1. In PET-negative individuals, the mean Aβ42 / Aβ40 ratio value was 0.184 with a standard deviation of 0.02, while in PET-positive individuals, the mean Aβ42 / Aβ40 ratio value was 0.152 with a standard deviation of 0.03 (p-value < 0.001). The receiver operating characteristic curve showed an area under the curve (AUC) of 0.8365 (Figure 2).

[0210] As explained in the Background section, the Aβ42 / Aβ40 concentration ratio has been used as a biomarker of cerebral amyloidosis in the early stages of Alzheimer's disease, where a decrease in Aβ42 concentration is observed in the presence of amyloid plaques. Thus, these results demonstrated that the method of the present invention can not only be used to monitor Aβ changes in response to treatment, but also for diagnostic purposes, as it allows for the differentiation of PET-positive and PET-negative individuals.

[0211] Example 6 Comparison of various combinations of SPE cartridges Based on the sample preparation for mass spectrometry described in Example 1 and the subsequent analysis described in Example 2, various SPE cartridge combinations were tested. The extracted chromatographic traces for Aβ40 and Aβ42 are shown in Figures 4-8. For comparison, the use of a single SPE step (mixed-mode reversed-phase-cation exchange) was also evaluated (Figure 3). The use of a single reversed-phase SPE was not evaluated because the resulting eluate was difficult to analyze successfully by micro-LC due to the duration of the chromatographic column.

[0212] Therefore, the following SPE combinations were evaluated: - Figure 4 shows the extracted chromatographic traces for Aβ40 (left) and Aβ42 (right) when a plasma sample was contacted with an acidic denaturant and separated first by reversed-phase mode SPE (HLB Prime) and then by a second mixed-mode reversed-phase-cation exchange SPE (MCX) according to protocol A. - Figure 6 shows the extracted chromatographic traces for Aβ40 (left) and Aβ42 (right) when a plasma sample was contacted with an acidic denaturant and separated first by reversed-phase mode SPE (HLB) and then by a second mixed-mode reversed-phase-cation exchange SPE (MCX) according to protocol B. - Figure 5 shows the extracted chromatographic traces for Aβ40 (left) and Aβ42 (right) when a plasma sample was contacted with an acidic denaturant and separated first by mixed-mode reversed-phase-cation exchange SPE (MCX) and then by a second reversed-phase mode SPE (HLB).

[0213] The figure clearly shows that the traces extracted for Aβ40 (left) and Aβ42 (right) when the first SPE is MCX followed by HLB are inferior to those obtained by using HLB or HLB Prime first followed by MCX. Furthermore, when compared to a single SPE step, the traces are much more intense, as is known in the art (Figure 3), indicating that the combination of two consecutive SPE steps improves the signal-to-noise ratio and therefore the sensitivity. 8 shows the chromatographic traces extracted for Aβ40 (left) and Aβ42 (right) when a plasma sample was contacted with an acidic denaturant and separated first by mixed-mode reversed-phase cation exchange SPE (MCX) and then by a second mixed-mode reversed-phase anion exchange SPE (MAX) (Protocol C). The traces are compared with those obtained when a plasma sample denatured with an acidic agent was separated first by reversed-phase SPE (HLB) and then by a second mixed-mode reversed-phase cation exchange SPE (MCX).

[0214] The chromatographic traces shown in Figure 8 show that using first an MCX SPE followed by a second MAX SPE similarly yields distinct peaks for Aβ40 and Aβ42, but this time they are not as intense as using first an HLB SPE followed by an MCX SPE. However, when the first SPE is replaced with HLB, the recovery of both analytes is very low. Figure 7 shows the chromatographic traces extracted for Aβ40 (left) and Aβ42 (right) when a plasma sample was contacted with a basic denaturing agent and separated first by mixed-mode reversed-phase anion exchange SPE (MAX) and then by a second mixed-mode reversed-phase cation exchange SPE (MCX) according to Protocol D. For comparison, Figure 7 also shows the traces obtained when a plasma sample denatured with a basic agent was separated first by reversed-phase SPE (HLB) and then by a second mixed-mode reversed-phase cation exchange SPE (MCX).

[0215] These results show that when plasma samples are denatured with a basic agent and then subjected to first MAX followed by MCX, the peaks obtained for Aβ40 and Aβ42 are almost as good as the combination of HLB followed by MCX when the samples are denatured with an acidic agent.

Claims

1. 1. A method for preparing a plasma sample containing amyloid beta peptides for analysis by mass spectrometry, comprising: a) contacting the plasma sample with a denaturing agent; b) subjecting the solution obtained in step a) to a first solid phase extraction step to recover a first eluate; c) performing a second solid phase extraction step on the first eluate obtained in step b) to recover a second eluate; and d) drying the second eluate obtained in step c) and processing it for analysis by mass spectrometry; A method characterized in that the sample obtained from step d) contains intact amyloid beta peptides Aβ40 and Aβ42.

2. 10. The method of claim 1, wherein the second solid phase extraction step is a cation exchange solid phase extraction.

3. 3. The method of claim 2, wherein the cation exchange solid phase extraction is strong cation exchange, weak cation exchange, or mixed mode reversed phase-cation exchange.

4. 10. The method of claim 1, wherein the second solid phase extraction step is anion exchange solid phase extraction.

5. 5. The method of claim 4, wherein the anion exchange solid phase extraction is strong anion exchange, weak anion exchange, or mixed mode reversed phase-anion exchange.

6. 6. The method according to claim 1, wherein in step a) the plasma sample is contacted with an acidic denaturing agent to obtain a solution having a pH of 4.5 or less.

7. 7. The method according to claim 6, wherein the acidic denaturant is an aqueous formic acid solution having a concentration of 40% to 70% (v / v).

8. 8. The method of claim 6, wherein the first solid phase extraction step is a reversed phase solid phase extraction.

9. 7. The method according to claim 4, wherein the first and second solid-phase extraction steps each comprise at least two washing steps, the first washing step of the first and second solid-phase extraction steps being carried out with a solution containing an acid, and the second washing step of the first and second solid-phase extraction steps being carried out with a solution containing a water-miscible polar organic solvent.

10. 10. The method of claim 9, wherein the acid-containing solution of the first washing step is the same as the acid-containing solution of the second washing step.

11. 10. The method of claim 9, wherein the acid-containing solution of the first cleaning step is different from the acid-containing solution of the second cleaning step.

12. 8. The method of any one of claims 1, 4 to 7, wherein the first solid phase extraction step is a cation exchange solid phase extraction.

13. 13. The method of claim 12, wherein the cation exchange solid phase extraction is strong cation exchange, weak cation exchange, or mixed mode reversed phase-cation exchange.

14. 14. The method according to claim 13, wherein the first and second solid-phase extraction steps each include at least two washing steps, the first washing step of the first solid-phase extraction being carried out with a solution containing an acid, the first washing step of the second solid-phase extraction being carried out with a solution containing a base, and the second washing steps of the first and second solid-phase extraction steps being carried out with a solution containing a water-miscible polar organic solvent.

15. 4. The method according to claim 1, wherein in step a) the plasma sample is contacted with a basic denaturing agent to obtain a solution having a pH of 11 or higher.

16. 16. The method of claim 15, wherein the basic denaturant is an aqueous solution of ammonium hydroxide at a concentration of 5 to 50% (v / v).

17. 17. The method of claim 15, wherein the first solid phase extraction step is anion exchange solid phase extraction.

18. 18. The method of claim 17, wherein the anion exchange solid phase extraction is strong anion exchange, weak anion exchange, or mixed mode reversed phase-anion exchange.

19. 19. The method according to claim 15, wherein the first and second solid-phase extraction steps each comprise at least two washing steps, the first washing step of the first solid-phase extraction being carried out with a solution containing a base, the first washing step of the second solid-phase extraction being carried out with a solution containing an acid, and the second washing steps of the first and second solid-phase extraction steps being carried out with a solution containing a water-miscible polar organic solvent.

20. 20. The method according to any one of claims 1 to 19, wherein the solution of step d) for treating the dried eluate is an aqueous solution comprising a surfactant and a reducing agent.

21. 21. The method according to claim 20, wherein the solution in step d) for treating the dried eluate is an aqueous solution containing Triton® X-100 at a concentration of 0.01% to 0.8% (v / v) and tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v).

22. 20. The method of claim 1, wherein the solution for treating the dried eluate is an aqueous solution comprising a surfactant, a reducing agent, a water-miscible polar organic solvent, and an acid.

23. 23. The method of claim 22, wherein the solution in step d) for treating the dried eluate is an aqueous solution containing Triton® X-100 at a concentration of 0.01% to 0.8% (v / v), tris-carboxyethylphosphine at a concentration of 0.1% to 0.2% (w / v), acetonitrile at a concentration of 3% to 7% (v / v), dimethylformamide at a concentration of 0.1% to 3% (v / v), and trifluoroacetic acid (TFA) at a concentration of 0.1% to 3% (v / v).

24. 24. The method of any one of claims 1 to 23, wherein the plasma sample is a human plasma sample.

25. 25. The method according to any one of claims 1 to 24, wherein the amount of plasma sample used in step a) is between 100 μL and 400 μL.

26. 26. The method according to any one of claims 1 to 25, characterized in that it does not involve immunoprecipitation or digestion of the plasma sample before analysis by mass spectrometry.

27. 27. The method according to any one of claims 1 to 26, characterized in that the processing of the eluate of step d) of claim 1 comprises adapting the eluate to liquid chromatography combined with mass spectrometry.

28. 28. A method for the mass spectrometric quantification of intact amyloid beta peptides Aβ40 and Aβ42 in a plasma sample, comprising steps a) to d) of the method according to any one of claims 1 to 27 for preparing the plasma sample, and further comprising: i) subjecting the solution obtained in step d) to a liquid chromatography step to separate the analytes of interest; ii) subjecting the analytes separated in step i) to ionization to produce one or more charged species; iii) separating said one or more charged species according to ion mobility; iv) detecting the one or more charged species separated in step iii) and determining their abundance by mass spectrometry; and v) determining the amount or concentration of intact amyloid beta peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged species measured in step iv) with a standard curve.

29. 29. The method of claim 28, wherein the liquid chromatography is micro-liquid chromatography (micro-HPLC), the ionization is electrospray ionization (ESI), the separation of the one or more charged species is performed by differential mobility spectrometry (DMS), and the mass spectrometry technique for detecting and measuring the abundance of the separated one or more charged species is multiple reaction monitoring (MRM) in a triple quadrupole instrument.

30. 30. The method of claim 28 or 29, wherein the standard curve is prepared with human plasma.

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