Methods for quantifying amyloid-β peptide in plasma by mass spectrometry

Abstract

This invention relates to a method for preparing a plasma sample containing amyloid β-peptides for mass spectrometry analysis, comprising the following steps: 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 eluent; c) performing a second solid-phase extraction step on the first eluent obtained in step b) to recover a second eluent; and d) drying the second eluent obtained in step c) and processing it for mass spectrometry analysis, wherein the solution obtained in step d) contains intact amyloid β-peptides Aβ40 and Aβ42.

Description

[0001] describe

[0002] This invention relates to the fields of medical and veterinary science for diagnosing amyloid diseases, and particularly to analytical methods for preparing plasma samples for the detection and quantification of amyloid Aβ40 and Aβ42 peptides by mass spectrometry.

[0003] background

[0004] Alzheimer's disease (AD) is a leading cause of dementia, characterized by a progressive neurodegenerative disease affecting 17% of people aged 75–84 and 32% of those over 85 (Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, Jan 2020, 194–208). AD is characterized by the gradual appearance of amyloid plaques in the brain, with a central core of amyloid deposits primarily composed of protofibrils of 40–42 amino acid peptides. These amyloid deposits are formed following the proteolytic processing of amyloid precursor protein (APP), resulting in the formation of insoluble Aβ peptides, primarily Aβ1–40 (Aβ40) and Aβ1–42 (Aβ42). In healthy patients, these peptides are cleared into the cerebrospinal fluid (CSF) or transported across the blood-brain barrier into the bloodstream. However, excessive production of amyloid peptides or reduced clearance of amyloid peptides leads to the formation of amyloid plaques characteristic of AD. These plaques primarily contain Aβ42 and act as a "sink" for peptides, reducing the concentration of Aβ42 in both CSF and blood.

[0005] Therefore, the Aβ42 / Aβ40 ratio is currently used as a biomarker for cerebral amyloidosis in the early stages of Alzheimer's disease, for inclusion in clinical trials. Aβ42 and Aβ40 can be measured by mass spectrometry (MS) or immunoassay, which are currently the main methods available for measurement in cerebrospinal fluid (CSF), where lower Aβ42 concentrations have been found in the presence of amyloid plaques (Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, Jan 2020, 194-208). However, CSF collection is quite invasive and requires specialized medical skills, making it less convenient for large-scale screening studies. Therefore, methods for quantifying plasma Aβ42 and Aβ40 are of great significance for diagnosing individuals with or without symptoms (Fandos et al., Plasma amyloidβ42 / 40 ratios as biomarkers for amyloidβcerebral deposition in cognitively normal individuals. Alzheimer's Dement, 2017 Sep 12; 8:179-187).

[0006] However, measuring the concentrations of Aβ42 and Aβ40 in plasma also presents several challenges. First, blood is a highly complex matrix containing a large number of different proteins, resulting in a total protein content in plasma that is 60 times higher than in CSF. Second, the concentrations of Aβ42 and Aβ40 are lower in plasma than in CSF due to their transport from the central nervous system to venous blood. Furthermore, the difference in plasma Aβ42 concentration between amyloid-positive and amyloid-negative individuals is smaller than the difference in CSF (Bateman and Budelier, Biomarkers of Alzheimer Disease, Journal of Applied Laboratory Medicine, Jan 2020, 194-208). For these reasons, the analysis of amyloid β-peptides in plasma is more challenging than that in CSF, and therefore requires more sensitive and accurate methods.

[0007] Previous studies using enzyme-linked immunosorbent assay (ELISA) to measure plasma amyloid beta-peptide concentrations have shown conflicting results regarding the Aβ42 / Aβ40 ratio in AD patients and healthy controls (Fukumoto et al., Age butnot diagnosis is the main predictor of plasma amyloid beta-protein levels, Arch Neurol. 2003; 60:958–964; Pérez-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). In fact, the mean difference between groups (i.e., healthy controls versus individuals with mild cognitive impairment) is as low as 10%–15%, on the same order of magnitude as the acceptable variability of the analytical method's accuracy and precision. This means that to detect such low inter-group differences, the variability of the analytical method must be well below 15%.

[0008] On the other hand, available mass spectrometry (MS) methods have been shown to be more sensitive and accurate than immunoassays. Currently, two MS-based analytical methods are known in the art for determining Aβ40 and Aβ42 in human plasma. The first method was published by Randall Bateman's group in 2017 (Ovod et al., Amyloid B concentrations and stable isotopelabelling kinetics of human plasma specific to central nervous system amyloidosis, Alzheimer's and Dementia, 2017 Oct; 13(10):1185). This method combines sample preparation by immunoprecipitation and Lys-N digestion with nanoLC-MS / MS. The second method was published by Akinori Nakamura's group in 2018 (Nakamura et al., High performance plasma amyloid-β biomarkers for Alzheimer's disease, Nature 2018 Feb 8; 554(7691):249-254) and combined sample preparation by double immunoprecipitation with MALDI-TOF / MS.

[0009] However, both methods are time-consuming and resource-intensive due to the need for expensive antibodies during immunoprecipitation. Furthermore, Bateman's method requires enzymatic digestion of the sample with the analyte, resulting in the detection of a mixture of N-truncated Aβ material rather than the complete Aβ peptide.

[0010] Therefore, there remains a need in the field for a sensitive and reproducible method for detecting and quantifying amyloid-β peptides in plasma samples that can be applied to large-scale screening studies.

[0011] Through extensive and in-depth experimentation, the inventors of this invention have surprisingly discovered a novel method for preparing plasma samples containing amyloid peptides, which allows for the accurate quantification of intact amyloid peptides Aβ40 and Aβ42 via mass spectrometry. Therefore, this new method reduces the variability of the Aβ42 / Aβ40 ratio measurement to a value lower than the actual difference between healthy controls and individuals with mild cognitive impairment.

[0012] Furthermore, the method of the present invention is carried out without the use of immunoprecipitation or digestion of the sample (essential steps in methods known in the art), and therefore provides a simpler and faster sample preparation, reducing cost and time requirements compared to currently available methods.

[0013] Finally, the method of the present invention complies with current FDA recommendations regarding the validation of bioanalytical methods and can therefore be applied to the diagnosis and / or differentiation of different stages of neurodegenerative diseases such as Alzheimer's disease.

[0014] Overview

[0015] In one aspect, the present invention relates to a method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, characterized by comprising the following steps:

[0016] a) Contact the plasma sample with a denaturing agent.

[0017] b) Perform a first solid-phase extraction on the solution obtained in step a) to recover the first eluent.

[0018] c) Perform a second solid-phase extraction step on the first eluent obtained in step b) to recover the second eluent, and

[0019] d) Dry the second eluent obtained in step c) and process it for mass spectrometry analysis.

[0020] The sample obtained from step d) contains intact amyloid β peptides Aβ40 and Aβ42.

[0021] In one embodiment of the method, the second solid-phase extraction step is cation exchange solid-phase extraction.

[0022] In another embodiment, cation exchange solid-phase extraction is a strong, weak, or mixed-mode reversed-phase cation exchange.

[0023] In another embodiment, the second solid-phase extraction step is anion exchange solid-phase extraction.

[0024] In another embodiment, the second anion exchange solid-phase extraction is a strong, weak, or mixed-mode reverse anion exchange.

[0025] In another embodiment, in step a), the plasma sample is contacted with an acidic denaturing agent to obtain a solution with a pH of less than or equal to 4.5.

[0026] In another embodiment, the acid denaturant is a solution of formic acid in water with a concentration between 40% (v / v) and 70% (v / v).

[0027] In another implementation, the first solid-phase extraction step is reversed-phase solid-phase extraction.

[0028] In another embodiment, each of the first and second solid-phase extraction steps includes at least two washing steps, characterized in that 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.

[0029] In another embodiment, the acid-containing solution in the first washing step is different from the acid-containing solution in the second washing step.

[0030] In another embodiment, the acid-containing solution in the first washing step is the same as the acid-containing solution in the second washing step.

[0031] In another embodiment, the first solid-phase extraction step is cation exchange solid-phase extraction.

[0032] In another embodiment, the first cation exchange solid-phase extraction is a strong, weak, or mixed-mode reversed-phase cation exchange.

[0033] In another embodiment, each of the first and second solid-phase extraction steps includes at least two washing steps, characterized in that the first washing step of the first solid-phase extraction is performed with a solution containing an acid, and the first washing step of the second solid-phase extraction is performed with a solution containing an alkali, 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.

[0034] In another embodiment, in step a), the plasma sample is contacted with an alkaline denaturant to obtain a solution having a pH greater than or equal to about 11.

[0035] In another embodiment, the alkaline denaturant is a solution of ammonium hydroxide in water with a concentration between 5% (v / v) and 50% (v / v).

[0036] In another embodiment, the first solid-phase extraction step is anion-exchange solid-phase extraction.

[0037] In another embodiment, the first anion exchange solid-phase extraction is a strong, weak, or mixed-mode reverse anion exchange.

[0038] In another embodiment, each of the first and second solid-phase extraction steps includes at least two washing steps, characterized in that the first washing step of the first solid-phase extraction is performed with a solution containing an alkali, and the first washing step of the second solid-phase extraction 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.

[0039] In yet another embodiment, the solution used to treat the second eluent for mass spectrometry analysis is an aqueous solution containing a surfactant and a reducing agent. In a preferred embodiment, the solution used in step d) to treat the dried eluent is an aqueous solution containing Triton X-100 at concentrations between 0.01% (v / v) and 0.8% (v / v) and tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v).

[0040] In another embodiment, the solution for treating the dried eluent in step d) is an aqueous solution comprising a surfactant, a reducing agent, a water-miscible polar organic solvent, and an acid. In a preferred embodiment, the solution for treating the dried eluent in step d) is an aqueous solution comprising Triton X-100 at concentrations between 0.01% (v / v) and 0.8% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), dimethylformamide at concentrations between 0.1% (v / v) and 3% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v).

[0041] In some embodiments of the 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 invention is between 100 μL and 400 μL.

[0042] In some embodiments of the present invention, the method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis does not include immunoprecipitation or digestion of the plasma sample prior to mass spectrometry analysis.

[0043] In a second aspect, the present invention relates to a method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry, characterized in that the method comprises steps a) to d) of the method for preparing a plasma sample as described herein, and further comprises the following steps:

[0044] i) Perform liquid chromatography on the solution obtained in step d) to separate the analyte of interest.

[0045] ii) Ionize the analytes separated in step i) to produce one or more charged substances;

[0046] iii) Separate the one or more charged substances based on the ion mobility of the one or more charged substances.

[0047] iv) Detect one or more of the charged substances separated in step iii) and measure their abundance by mass spectrometry; and

[0048] v) Determine the amount or concentration of intact amyloid β-peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged substances measured in step iv) with a standard curve.

[0049] In some embodiments, the method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in plasma samples by mass spectrometry is characterized in that the liquid chromatography is micro-HPLC, the ionization is electrospray ionization (ESI), the separation of one or more charged substances is performed by differential mobility spectrometry (DMS), and the mass spectrometry technique used to detect and measure the abundance of the separated one or more charged substances is multiple reaction monitoring (MRM) in a triple quadrupole.

[0050] In other embodiments of the invention, the standard curve used in the method for quantifying intact amyloid β peptides Aβ40 and Aβ42 in plasma samples by mass spectrometry is prepared from human plasma.

[0051] In a third aspect, the present invention relates to an aqueous solution for treating dried eluents for mass spectrometry analysis, the aqueous solution comprising Triton X-100 at concentrations between 0.01% (v / v) and 0.8% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), dimethylformamide at concentrations between 0.1% (v / v) and 3% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v). Brief description of the attached diagram

[0053] Figure 1 A graph is shown representing the Aβ42 / Aβ40 ratios of 36 individuals (PET-negative or PET-positive) quantified by the method of the present invention.

[0054] Figure 2 Showing the target Figure 1 A graph of the ROC curve calculated from the Aβ42 / Aβ40 ratio.

[0055] Figure 3 The chromatograms obtained from plasma samples undergoing a single SPE step (mixed-mode reversed-phase cation exchange (MCX)) are shown. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0056] Figure 4 The chromatograms obtained from a plasma sample following Protocol A, which underwent a first SPE step (reversed phase (HLB prime)) followed by a second SPE MCX, are shown. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0057] Figure 5 The chromatograms obtained from plasma samples that underwent the first SPE step MCX and then the second SPE HLB are shown. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0058] Figure 6 The chromatograms obtained from plasma samples undergoing the first SPE step (HLB) followed by the second SPE step (MCX) according to Protocol B are shown. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0059] Figure 7 The chromatograms obtained from plasma samples undergoing a first SPE step (mixed-mode reversed-phase anion exchange SPE(MAX)) followed by a second SPE MCX according to scheme D are shown, compared to a combination that undergoes a first HLB step followed by a second MCX step. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0060] Figure 8 The chromatograms obtained from plasma samples undergoing the first SPE step (MCX) followed by the second SPE step (MAX) according to Protocol C are shown, compared to the combination of undergoing the first HLB step followed by the second MCX step. The trace in the left chromatogram relates to Aβ40, and the trace in the right chromatogram relates to Aβ42.

[0061] Detailed description

[0062] The following description is merely illustrative of various embodiments of the invention. Therefore, the specific modifications discussed are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the spirit or scope of the subject matter presented herein, and it should be understood that such equivalent embodiments will be included herein.

[0063] As used in this invention, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly specifies otherwise.

[0064] Throughout this specification and claims, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” shall be understood to mean including the stated element or group of elements, but not excluding any other element or group of elements.

[0065] The terms “analyte,” “substance,” “sample,” “component,” “chemical substance,” and “ion” can all be used herein to refer to substances that are to be analyzed, identified, and quantified by the methods of the present invention.

[0066] As used herein, the terms “solid phase extraction” or “SPE” refer to the process by which a mixture is separated into components. These components are dissolved and / or suspended in a solution (“sample solution”) and are separated from each other by their different affinities for a solid (“stationary phase”) through which the solution passes. In some cases, unwanted components in the sample solution may be retained by the stationary phase as the sample solution passes through it (i.e., the analyte in the sample solution is purified). In other cases, desired components may be retained by the stationary phase (i.e., the analyte of interest is retained in the stationary phase), and a second mobile phase is used to elute the analyte retained in the stationary phase for further processing or analysis. The “stationary phase” is typically contained in a “cartridge,” “tip,” or “column,” which can be aggregated in a multi-well plate, which is particularly convenient for large screening studies. Solid phase extraction cartridges, columns, tips, and multi-well plates are commercially available or can be prepared according to methods known in the art.

[0067] The term "purification" refers to the procedure of enriching one or more analytes of interest in a quantity relative to other components in a sample that may interfere with the detection of the analyte of interest. As used herein, the term "purification" does not mean the removal of all substances other than the analyte of interest from a sample.

[0068] "Immunoprecipitation" refers to a purification procedure that uses antibodies (including polyclonal or monoclonal antibodies) to enrich one or more analytes of interest in a sample.

[0069] As used herein, the term “digestion” generally refers to any suitable method for degrading or cleaving peptides or proteins, including, for example, the use of cellular enzymes (proteases) and intramolecular digestion.

[0070] As used herein, the terms “mass spectrometry” or “MS” refer to an analytical technique that measures the mass-to-charge ratio of a particular analyte. MS is widely used to encompass all components and systems that can be used to detect and identify analytes using their mass-to-charge ratio. MS techniques typically involve ionizing the analyte (although they may have previously 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. Analytes can be ionized and detected by any suitable method.

[0071] The term "chromatography" refers to a process by which a mixture carried by a liquid or gas is separated into components that are eluted at different residence times due to the differential distribution of chemical entities as they flow through a stationary phase. Therefore, "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 moves through a column. The separation is due to the distribution of the components of the mixture between one or more stationary and mobile phases. Examples of LC or HPLC include normal-phase liquid chromatography (NPLC), reversed-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.

[0072] The terms “micro-HPLC” or “micro-LC” refer to high-performance liquid chromatography (HPLC) using low flow rates (i.e., 1-25 μl / min) and capillary columns (inner diameter 150-500 μm).

[0073] As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions with a net charge equal to one or more charge units. Negative ions have a net negative charge, and positive ions have a net positive charge. Non-limiting examples of ionization include electron ionization, chemical ionization, electrospray ionization (ESI), pressure photoionization (APPI), matrix-assisted laser desorption / ionization (MALDI), and pressure chemical ionization (APCI).

[0074] "Electrospray ionization" or "ESI" refers to an ionization method that transfers analytes of interest into the gas phase by means of ionization, in which a sample solution containing the analyte of interest is sprayed into an electric field to form charged droplets.

[0075] The term "ion mobility spectrometry (IMS)" refers to an analytical technique used to separate and identify molecules based on their migration rates in a carrier buffer gas. The term "differential mobility spectrometry (DMS)" refers to a specific type of IMS that separates ionized molecules based on the differences in ion mobility between high and low electric fields, and in gases at or near atmospheric pressure.

[0076] The term “multiple reaction monitoring (MRM),” also known as “selected reaction monitoring (SRM),” refers to a scanning mode in a tandem MS in which two (or more) analytical devices (i.e., quadrupoles) are tuned to monitor one or more selected parent-product pairs of the analyte of interest.

[0077] The terms "tandem mass spectrometry" or "MS / MS" refer to mass spectrometry that performs more than one stage of mass analysis, where the more than one stage is separated in time or space. For example, time-tandem mass spectrometry may include a single mass analyzer (e.g., an ion trap) where specific ions are first captured, separated, and fragmented, and then the fragments are analyzed in the same mass analyzer. Spatial tandem mass spectrometry includes more than one analyzer. The analyzers are separated by one or more reaction zones (i.e., collision cells filled with gases such as argon, xenon, nitrogen, or helium) where the analyte dissociates. Finally, the fragmented ions are filtered in a final analyzer and then detected. Typically, two analyzers are used. These analyzers may be of the same type or may not be of the same type.

[0078] As used herein, the term “ROC” stands for “receiver operating characteristic.” ROC analysis can be used to evaluate the diagnostic performance or predictive ability of a test or analytical method. An ROC plot is a graph of the sensitivity and specificity of a test at different thresholds or cutoff values. Each point on the ROC curve represents the sensitivity and its corresponding specificity. Thresholds can be selected based on the ROC curve to identify points where both sensitivity and specificity are acceptable, and these values ​​can be used when applying the test for diagnostic purposes. If only specificity is optimized, the test will be less likely to produce false positives (diagnosing disease in more subjects without the disease), at the cost of an increased likelihood that some disease cases will not be identified (e.g., false negatives). If only sensitivity is optimized, the test will be more likely to identify most or all subjects with the disease, but will also diagnose disease in more subjects without the disease (e.g., false positives). Users can modify parameters in a manner readily understood by those skilled in the art, and thus select ROC thresholds appropriate for specific clinical situations.

[0079] The term "area under the curve (AUC)" quantifies the overall ability of a test to distinguish between different sample properties (in this case, between those with Aβ amyloidosis (i.e., amyloid-positive) and those without Aβ amyloidosis (i.e., amyloid-negative)). A test that is not superior to random probability in identifying true positives will produce an AUC of 0.5. A test with perfect specificity and sensitivity (i.e., producing no false positives or false negatives) will have an AUC of 1.00.

[0080] 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 pertains. Exemplary methods and materials are described below, although similar or equivalent methods and materials may also be used, and will be apparent to those skilled in the art.

[0081] Unless otherwise expressly stated, each embodiment in this specification, with necessary modifications, will be applicable to every other embodiment.

[0082] This invention relates to a method for preparing plasma samples containing amyloid β-peptide for mass spectrometry analysis.

[0083] Amyloid β peptide (also known as Aβ or Abeta peptide) is a peptide derived from amyloid precursor protein (APP) through proteolytic processing. As used herein, the term "amyloid β" refers to total amyloid β (Aβ) protein, Aβ40, Aβ42, or another Aβ subtype. In some embodiments, the sample may contain Aβ40. In other embodiments, the sample may contain Aβ42. In a preferred embodiment, the sample may contain both Aβ40 and Aβ42.

[0084] As used herein, the term "intact" amyloid β refers to the full-length peptide that has not undergone chemical / enzymatic cleavage or any other peptide modification. In the exact cases of Aβ40 and Aβ42 peptides, the intact amyloid β peptide Aβ42 refers to the 42 amino acids corresponding to amino acids 672 to 713 of human APP subtype 770 (typically, UniProtKB accession number P05067), and the intact amyloid β peptide Aβ40 refers to the 40 amino acids corresponding to amino acids 672 to 711 of human APP subtype 770 (typically, UniProtKB accession number P05067).

[0085] In some embodiments, the sample is a plasma sample derived from the subject. Suitable subjects include humans or any other mammals, livestock (such as pigs, cattle, 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 zoological animals. In a preferred embodiment, the subject is a mammal. In a more preferred embodiment, the subject is a human.

[0086] Plasma samples can be used "as is," or protein fractions can be separated from the plasma sample using standard techniques. For example, plasma samples can be concentrated, diluted, or extracted. Suitable extraction techniques may include surfactants, acids, bases, organic solvents, or other methods known in the art. In some embodiments, the original sample may be pretreated to reduce matrix complexity. In some embodiments, these pretreatments are techniques well known to those skilled in the art, such as liquid-liquid extraction or protein precipitation, but other techniques known to those skilled 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.

[0087] In a first aspect, the present invention relates to a method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis. The method includes the following steps:

[0088] a) Contact the plasma sample with a denaturing agent.

[0089] b) Perform a first solid-phase extraction on the solution obtained in step a) to recover the first eluent.

[0090] c) Perform a second solid-phase extraction step on the first eluent obtained in step b) to recover the second eluent, and

[0091] d) Dry the second eluent obtained in step c) and process it for mass spectrometry analysis.

[0092] The sample obtained from step d) contains intact amyloid β peptides Aβ40 and Aβ42.

[0093] The term “processing” in step d) means making the sample suitable for mass spectrometry analysis, and includes, for example, making the sample suitable for liquid chromatography coupled with mass spectrometry.

[0094] In some embodiments, a plasma sample containing amyloid β-peptide is contacted with an acidic denaturing agent. In a more preferred embodiment, the acidic denaturing agent is an organic acid. In yet more preferred embodiments, the organic acid is a carboxylic acid. In still more preferred embodiments, the carboxylic acid is a monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤6, for example, about ≤5, such as about ≤4. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤4. In a preferred embodiment, the monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid is a C1-C10 haloalkylcarboxylic acid, a C1-C10 alkylcarboxylic acid, or a combination thereof. For example, the carboxylic acid may be selected from C1-C5 haloalkyl monocarboxylic acids, C1-C5 alkyl monocarboxylic acids, or a combination thereof. In yet more preferred embodiments, the carboxylic acid is formic acid.

[0095] All pKa values ​​disclosed in this paper were measured in water at 25°C and 1 atmosphere.

[0096] Therefore, in some embodiments, the acid denaturing agent is formic acid. In a more preferred embodiment, the acid denaturing agent is a solution of formic acid in water containing between 40% (v / v) and 70% (v / v) of formic acid. In a preferred embodiment, the formic acid solution contains between 45% (v / v) and 60% (v / v) of formic acid in water. In a more preferred embodiment, the formic acid solution contains approximately 50% (v / v) of formic acid in water.

[0097] In some preferred embodiments, a plasma sample containing amyloid β-peptide is contacted with an acidic denaturing agent to obtain a solution having a pH of less than or equal to about 4.5. In a more preferred embodiment, the pH of the solution can be between about 0.1 and about 4.5. For example, the pH of the solution can be between about 0.4 and about 3. The pH of the solution can be between about 0.5 and 2, for example, between about 0.8 and 1.5. In a more preferred embodiment, the solution obtained after contacting the plasma sample with the acidic denaturing agent has a pH between 1 and 1.4, more preferably between 1.1 and 1.3, and even more preferably about 1.2.

[0098] The volume of the acidic denaturing agent used in the method of the present invention depends on the volume of the plasma sample used and the pH of the acidic denaturing agent. Those skilled in the art can determine the volume of the acidic denaturing agent through simple calculations to obtain a solution with a specific pH. In some preferred embodiments of the invention, the acidic denaturing agent is formic acid with a concentration in water between 40% (v / v) and 70% (v / v). In a more preferred embodiment of the invention, a plasma sample with a volume between 100 μl and 400 μl is contacted with formic acid with a volume between 200 μl and 800 μl and a concentration in water between 40% (v / v) and 70% (v / v).

[0099] Using formic acid as a denaturant to denature the plasma samples of the present invention provides several advantages, such as disrupting many Aβ-plasma protein (i.e. IgG) interactions while keeping the Aβ peptide in solution (without precipitation).

[0100] In some embodiments, a plasma sample containing amyloid β-peptide is contacted with a basic denaturing agent. In a more preferred embodiment, the basic denaturing agent is 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. In yet more preferred embodiments, the basic denaturing agent is a solution of ammonium hydroxide in water containing between 15% (v / v) and 40% (v / v) of ammonium hydroxide. In a preferred embodiment, the solution of the basic agent contains between 20% (v / v) and 50% (v / v) of ammonium hydroxide in water. In a more preferred embodiment, the solution of the basic agent contains about 25% (v / v) of ammonium hydroxide in water.

[0101] In some preferred embodiments, a plasma sample containing amyloid β-peptide is contacted with an alkaline denaturing agent to obtain a solution having a pH greater than or equal to about 11. In a more preferred embodiment, the pH of the solution can be between about 11 and about 13. For example, the pH of the solution can be between about 11 and about 12. The pH of the solution can be between about 11 and 11.5, most preferably about 11.3.

[0102] The volume of the basic denaturing agent used in the method of the present invention depends on the volume of the plasma sample used and the pH of the basic denaturing agent. Those skilled in the art can determine the volume of the basic denaturing agent through simple calculations to obtain a solution with a specific pH. In some preferred embodiments of the invention, the basic denaturing agent is ammonium hydroxide with a concentration in water between 15% (v / v) and 40% (v / v). In a more preferred embodiment of the invention, a plasma sample with a volume between 100 μl and 400 μl is contacted with ammonium hydroxide with a volume between 200 μl and 800 μl and a concentration in water between 15% (v / v) and 40% (v / v).

[0103] Using ammonium hydroxide as a denaturant to denature the plasma samples of the present invention provides several advantages, such as disrupting many Aβ-plasma protein (i.e. IgG) interactions while keeping the Aβ peptide in solution (without precipitation).

[0104] After the sample denatured, the amyloid β-peptide was separated from the other components of the sample by solid phase extraction (SPE).

[0105] Several types of SPEs are known in the art. They can be classified according to the chemical properties of the stationary phase used. Thus, in normal-phase SPEs, the stationary phase is more polar than the mobile phase, such as silica gel or alumina, as a combination of a stationary phase and a mobile phase containing a less polar eluent (i.e., hexane). Conversely, reverse-phase SPEs use low-polarity fillers, such as octadecylsilane or octylsilane, bonded to silica or polymer microbeads, and the mobile phase is typically a mixture of water and organically miscible solvents and modifiers. On the other hand, during ion-exchange SPEs, components are separated based on electrostatic interactions between the components and positively or negatively charged groups on the stationary phase. Thus, ion-exchange SPEs include anion-exchange SPEs and cation-exchange SPEs. In anion-exchange SPEs, the stationary phase contains positively charged groups that interact with and retain negatively charged anions (such as acids), while in cation-exchange SPEs, the stationary phase contains negatively charged groups that interact with and retain positively charged cations (such as bases). Strong cation exchange adsorbents contain aliphatic sulfonic acid groups that are always negatively charged in aqueous solutions, while weak cation exchange adsorbents contain aliphatic carboxylic acids that are charged when the pH is above 5. More than one residence mechanism can also be combined in the same column, a so-called mixed-mode SPE, which typically combines reversed-phase and ion exchange columns. Therefore, mixed-mode reversed-phase anion exchange and mixed-mode reversed-phase cation exchange are known SPEs in the art.

[0106] The purpose of solid-phase extraction in the method of this invention is to separate and discard unwanted components from plasma samples in order to purify and concentrate the amyloid β-peptide of interest in the sample.

[0107] In some embodiments, the method of the present invention includes a first solid-phase extraction step on the solution obtained after contacting the sample with a denaturing agent. In a preferred embodiment, the method of the present invention includes a second solid-phase extraction step after the first solid-phase extraction step. Therefore, in some embodiments, the method of the present invention for preparing a sample containing amyloid β-peptide for mass spectrometry analysis includes two consecutive solid-phase extraction steps.

[0108] In some embodiments, the first and second solid-phase extraction steps of the method of the present invention for preparing a sample containing amyloid β-peptide can be of any type known to those skilled in the art.

[0109] In some embodiments, the first solid-phase extraction step is a reversed-phase SPE. In some embodiments, the first solid-phase extraction step is a cation exchange SPE. In some embodiments, the first solid-phase extraction step is an anion exchange SPE. In some embodiments, the cation exchange SPE is a strong, weak, or mixed-mode reversed-phase cation exchange. In some embodiments, the anion exchange SPE is a strong, weak, or mixed-mode reversed-phase anion exchange.

[0110] In some embodiments, the second solid-phase extraction step is a reversed-phase SPE. In some embodiments, the second solid-phase extraction step is a cation exchange SPE. In some embodiments, the second solid-phase extraction step is an anion exchange SPE. In some embodiments, the cation exchange SPE is a strong, weak, or mixed-mode reversed-phase cation exchange. In some embodiments, the anion exchange SPE is a strong, weak, or mixed-mode reversed-phase anion exchange.

[0111] In some embodiments, the first solid-phase extraction step is a reversed-phase SPE, and the second solid-phase extraction step is a cation exchange SPE, more preferably the cation exchange SPE is a strong, weak, or mixed-mode reversed-phase cation exchange.

[0112] In some embodiments, the first solid-phase extraction step is a reversed-phase SPE, and the second solid-phase extraction step is an anion exchange SPE, more preferably the anion exchange SPE is a strong, weak or mixed-mode reversed-phase anion exchange.

[0113] In some embodiments, the first solid-phase extraction step is a cation exchange SPE, more preferably a strong, weak, or mixed-mode reversed-phase cation exchange, and the second solid-phase extraction step is an anion exchange SPE, more preferably a strong, weak, or mixed-mode reversed-phase anion exchange.

[0114] In some embodiments, the first solid-phase extraction step is an anion exchange SPE, more preferably the anion exchange SPE is a strong, weak or mixed-mode reversed-phase anion exchange, and the second solid-phase extraction step is a cation exchange SPE, more preferably the cation exchange SPE is a strong, weak or mixed-mode reversed-phase cation exchange.

[0115] In some preferred embodiments of the method of the present invention for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, as disclosed herein, the plasma sample is contacted with an acidic denaturing agent, followed by a first SPE and a second SPE, wherein the first SPE is a reversed-phase SPE and the second solid-phase extraction step is a cation exchange SPE.

[0116] In some preferred embodiments of the method of the present invention for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, as disclosed herein, the plasma sample is contacted with an acidic denaturing agent, followed by a first SPE and a second SPE, wherein the first SPE is a reversed-phase SPE and the second solid-phase extraction step is an anion-exchange SPE.

[0117] In some preferred embodiments of the method of the present invention for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, as disclosed herein, the plasma sample is contacted with an acidic denaturing agent, followed by a first SPE and a second SPE, wherein the first SPE is a cation exchange SPE and the second solid-phase extraction step is an anion exchange SPE.

[0118] In some preferred embodiments of the method of the present invention for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, as disclosed herein, the plasma sample is contacted with an alkaline denaturant, followed by a first SPE and a second SPE, wherein the first SPE is an anion exchange SPE and the second solid-phase extraction step is a cation exchange SPE.

[0119] According to the method of the present invention, after denaturing the protein in a plasma sample containing amyloid peptides, a specific combination of two consecutive SPE steps as described herein allows for the purification of intact Aβ40 and Aβ42 peptides, which can be further analyzed by mass spectrometry without any additional purification steps.

[0120] The scheme for performing the first and second SPE steps of the present invention includes conditioning and equilibration of the stationary phase, loading the sample into a column or microcolumn, at least one washing step, and at least one elution step. These steps are well known to those skilled in the art, and the specific conditions for each step are also well known in the art.

[0121] In one embodiment of the invention, the methods for performing the first and second SPE steps of the invention are those known in the art. In another embodiment of the invention, the first and second SPE steps include at least one washing step to remove unwanted components from the sample. In a preferred embodiment of the invention, each of the first and second SPE steps includes at least two washing steps.

[0122] Suitable washing solutions are known in the art. In some embodiments, the washing 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 washing 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 categories 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 every member of the above categories is water-miscible; however, those skilled in the art can readily determine that members of a particular category are water-miscible. In yet more preferred embodiments, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof. In a particularly preferred embodiment, the water-miscible organic solvent for the washing solution of the SPE step of the method of the present invention is acetonitrile. In other embodiments, the washing solution used in the SPE step of the method of the present invention is a solution of an alkali (such as a water-soluble hydroxide, carbonate, oxide, or combination thereof). In a preferred embodiment, the washing solution used in the SPE step of the method of the present invention is a solution of ammonium hydroxide.

[0123] In some preferred embodiments, the washing solution used for the first wash is trifluoroacetic acid (TFA). During the SPE step of the present invention, the concentration of TFA that can be used for the first wash is between 0.01% (v / v) and 10% (v / v) of TFA in water. In another preferred embodiment, the washing solution is between 0.05% (v / v) and 1% (v / v) of TFA in water. In yet another more preferred embodiment, the washing solution is about 0.1% (v / v) of TFA in water. In a preferred embodiment, during the first SPE step of the present invention, the washing solution used for the first wash is between 0.05% (v / v) and 1% (v / v) of TFA in water, more preferably about 0.1% of TFA in water.

[0124] In some preferred embodiments, the washing solution used for the first wash is formic acid. During the SPE step of the present invention, the concentration of formic acid that can be used for the first wash is between 15% (v / v) and 35% (v / v) formic acid in water. In a preferred embodiment, the washing solution is between 20% (v / v) and 30% (v / v) formic acid in water. In a more preferred embodiment, the washing solution is about 25% (v / v) formic acid in water. In another preferred embodiment, the washing solution used for the first wash during the second SPE step of the present invention is between 20% (v / v) and 30% (v / v) formic acid in water, more preferably about 25% (v / v) formic acid in water.

[0125] In some preferred embodiments, the washing solution used for the first wash is an ammonium hydroxide solution. During the SPE step of the invention, the concentration of ammonium hydroxide used for the first wash can be between 2% and 50% (v / v) of ammonium hydroxide in water. In a preferred embodiment, the washing solution is between 2% (v / v) and 30% (v / v) of ammonium hydroxide in water. In a more preferred embodiment, the washing solution is about 10% (v / v) of ammonium hydroxide in water. In another preferred embodiment, the washing solution used for the first wash during the second SPE step of the invention is between 5% (v / v) and 30% (v / v) of ammonium hydroxide in water, more preferably about 10% (v / v) of ammonium hydroxide in water.

[0126] In some preferred embodiments, the washing solution used for the second wash is acetonitrile. During the SPE step of the present invention, the concentration of acetonitrile that can be used for the second wash is between 5% (v / v) and 80% (v / v) acetonitrile in water. In a preferred embodiment, the washing solution is between 10% (v / v) and 60% (v / v) acetonitrile in water.

[0127] In a preferred embodiment, during the first SPE step of the invention, the washing solution used for the second washing is acetonitrile in water at a concentration between 5% (v / v) and 15% (v / v). In a more preferred embodiment, the washing solution is about 10% (v / v) acetonitrile in water.

[0128] In a preferred embodiment, during the second SPE step of the invention, the washing solution used for the second washing is acetonitrile in water at a concentration between 40% (v / v) and 70% (v / v). In a more preferred embodiment, the washing solution is acetonitrile in water at a concentration of about 60% (v / v).

[0129] In other preferred embodiments, a third wash is performed during the first SPE step of the invention. In a preferred embodiment, the wash solution for the third wash comprises acetonitrile at a concentration between 90% (v / v) and 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).

[0130] In a preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing between 0.05% (v / v) and 1% (v / v) TFA in water, and the second washing step being performed with a solution containing between 5% (v / v) and 15% (v / v) acetonitrile in water. The second SPE step includes at least three washing steps, the first washing step being performed with a solution containing between 20% (v / v) and 30% (v / v) formic acid in water, the second washing step being performed with a solution containing between 40% (v / v) and 70% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing between 90% (v / v) and 100% (v / v) acetonitrile.

[0131] In a more preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing about 0.1% (v / v) TFA in water, and the second washing step being performed with a solution containing about 10% (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 about 25% (v / v) formic acid in water, the second washing step being performed with a solution containing about 60% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing about 100% (v / v) acetonitrile.

[0132] In a preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing between 0.05% (v / v) and 10% (v / v) methanol in water, and the second washing step being performed with a solution containing between 5% (v / v) and 15% (v / v) acetonitrile in water. The second SPE step includes at least three washing steps, the first washing step being performed with a solution containing between 0.5% (v / v) and 50% (v / v) formic acid in water, the second washing step being performed with a solution containing between 30% (v / v) and 70% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing between 90% (v / v) and 100% (v / v) acetonitrile.

[0133] In a preferred embodiment, the first SPE step includes at least three washing steps: the first washing step is performed with a solution containing between 5% (v / v) and 50% (v / v) formic acid in water; the second washing step is performed with a solution containing between 40% (v / v) and 70% (v / v) acetonitrile in water; and the third washing step is performed with a solution containing between 90% (v / v) and 100% (v / v) acetonitrile in water. The second SPE step includes at least two washing steps: the first washing step is performed with a solution containing between 0.5% (v / v) and 15% (v / v) ammonium hydroxide in water; and the second washing step is performed with a solution containing between 30% (v / v) and 70% (v / v) acetonitrile in water.

[0134] In a more preferred embodiment, the first SPE step includes at least three washing steps: the first washing step is performed with a solution containing about 25% (v / v) FA in water; the second washing step is performed with a solution containing about 60% (v / v) acetonitrile in water; and the third washing step is performed with a solution containing about 100% (v) acetonitrile. The second SPE step includes at least two washing steps: the first washing step is performed with a solution containing about 10% (v / v) ammonium hydroxide in water; and the second washing step is performed with a solution containing about 60% (v / v) acetonitrile in water.

[0135] In a more preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing between 1% (v / v) and 20% (v / v) of ammonium hydroxide in water, and the second washing step being performed with a solution containing between 30% (v / v) and 70% (v / v) of acetonitrile in water, and the second SPE step including at least three washing steps, the first washing step being performed with a solution containing between 0.5% (v / v) and 50% (v / v) of formic acid in water, the second washing step being performed with a solution containing between 30% (v / v) and 70% (v / v) of acetonitrile in water, and the third washing step being performed with a solution containing between 90% (v / v) and 100% (v / v) of acetonitrile.

[0136] In a more preferred embodiment, the first SPE step includes at least two washing steps, the first washing step being performed with a solution containing about 10% (v / v) ammonium hydroxide in water, and the second washing step being performed with a solution containing about 60% (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 about 25% (v / v) formic acid in water, the second washing step being performed with a solution containing about 60% (v / v) acetonitrile in water, and the third washing step being performed with a solution containing about 100% (v / v) acetonitrile.

[0137] Following at least one washing step performed during any SPE step, it is necessary to elute the analyte retained in the stationary phase. Suitable elution solutions are well known to those skilled in the art.

[0138] In some embodiments, the analyte retained in the stationary phase during the first SPE step is eluted with an elution solution containing a surfactant and a water-miscible polar organic solvent. In other embodiments, the analyte retained in the stationary phase during the first SPE step is eluted with an elution solution containing a base and a water-miscible polar organic solvent. In still other embodiments, the analyte retained in the stationary phase during the first SPE step is eluted with an elution solution containing an acid and a water-miscible polar organic solvent.

[0139] For example, water-miscible organic solvents can be polar aprotic organic solvents or protic organic solvents. Non-limiting examples of categories 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 every member of the above categories is water-miscible; however, those skilled in the art can readily determine that members of a particular category 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 eluent after the first SPE step of the method of the present invention is acetonitrile. The surfactant of the eluent can be of any type known to those skilled in the art, preferably nonionic. In some embodiments, the surfactant is nonionic. In some preferred embodiments, the nonionic surfactant is of the ethoxylated type. In a more preferred embodiment, the ethoxylated 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 in the elution solution can be an organic acid, preferably acetic acid, formic acid, or trifluoroacetic acid (TFA).

[0140] In a preferred embodiment, the eluent used after the first SPE step of the method of the present invention comprises Triton X-100 at a concentration between 1% (v / v) and 5% (v / v) and acetonitrile at a concentration between 20% (v / v) and 40% (v / v). In a more preferred embodiment, the eluent consists of about 2% (v / v) Triton X-100 and about 30% (v / v) acetonitrile in water.

[0141] In another preferred embodiment, the eluent used after the first SPE step of the method of the present invention comprises ammonium hydroxide at a concentration between 5% (v / v) and 15% (v / v) and acetonitrile at a concentration between 50% (v / v) and 90% (v / v). In a more preferred embodiment, the eluent consists of about 10% (v / v) ammonium hydroxide and about 75% (v / v) acetonitrile in water.

[0142] In another preferred embodiment, the eluent used after the first SPE step of the method of the present invention comprises trifluoroacetic acid at a concentration between 1% (v / v) and 15% (v / v) and acetonitrile at a concentration between 50% (v / v) and 90% (v / v). In a more preferred embodiment, the eluent consists of about 5% (v / v) trifluoroacetic acid and about 70% (v / v) acetonitrile in water.

[0143] In some embodiments, the analyte retained in the stationary phase during the second SPE step is eluted with an elution solution containing a polar organic solvent miscible with base and water. In other embodiments, the analyte retained in the stationary phase during the second SPE step is eluted with an elution solution containing a polar organic solvent miscible with acid and water.

[0144] 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 water-miscible organic solvent can be a polar aprotic organic solvent or a protic organic solvent. Non-limiting examples of categories 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 every member of the above categories is water-miscible; however, those skilled in the art can readily determine that members of a particular category are water-miscible. In a further 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 eluent after the second SPE step of the method of the present invention is acetonitrile. The acid in the eluent can be an organic acid, preferably acetic acid, formic acid, or trifluoroacetic acid (TFA).

[0145] In a preferred embodiment, the eluent used after the second SPE step of the method of the present invention comprises ammonium hydroxide at a concentration between 5% (v / v) and 15% (v / v) and acetonitrile at a concentration between 50% (v / v) and 90% (v / v). In a more preferred embodiment, the eluent consists of about 10% (v / v) ammonium hydroxide and about 75% (v / v) acetonitrile in water.

[0146] In another preferred embodiment, the eluent used after the first SPE step of the method of the present invention comprises trifluoroacetic acid at a concentration between 1% (v / v) and 15% (v / v) and acetonitrile at a concentration between 50% (v / v) and 90% (v / v). In a more preferred embodiment, the eluent consists of about 5% (v / v) trifluoroacetic acid and about 70% (v / v) acetonitrile in water.

[0147] After eluting the analyte of interest during the first SPE step of the present invention, the pH of the resulting eluent can be altered to prepare it for subsequent steps. In a preferred embodiment, the eluent obtained after elution during the first SPE is contacted with formic acid (preferably formic acid with a concentration in water between 30% (v / v) and 70% (v / v), more preferably formic acid with a concentration in water of about 50% (v / v)) to lower the pH of the solution containing the analyte of interest.

[0148] The eluent containing the purified, intact amyloid β-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 may include, but are not limited to, evaporation and lyophilization in a centrifugal vacuum concentrator (e.g., Thermo SpeedVac, Genevac). In a preferred embodiment, the eluent obtained after the second SPE step of the method of the present invention for preparing a sample containing amyloid β-peptide is dried in a vacuum concentrator at a temperature between 30°C and 50°C for at least 30 minutes. In a more preferred embodiment, the eluent is dried in a vacuum concentrator at a temperature between 40°C and 47°C for about 35 minutes.

[0149] After drying the eluent, the analyte needs to be resuspended in a solution suitable for a specific downstream assay. In some embodiments, the downstream assay may be an antibody-based detection method, such as ELISA, but in more preferred embodiments, the downstream assay is mass spectrometry, including liquid chromatography coupled to a mass spectrometer. Suitable solutions for different downstream assays are known in the art.

[0150] In some embodiments of the invention, the analyte is further subjected to mass spectrometry analysis, including liquid chromatography coupled to mass spectrometry, and the solution in which the dried analyte is resuspended contains 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 contains a surfactant and a reducing agent. In other embodiments, the solution in which the dried analyte is resuspended contains a surfactant, a reducing agent, a polar organic solvent, and an acid.

[0151] According to the present invention, the surfactant in the solution for resuspending the dried analyte can be of any type known to those skilled in the art, preferably nonionic. In some embodiments, the surfactant is nonionic. In some preferred embodiments, the nonionic surfactant is an ethoxylated type. In yet more preferred embodiments, the ethoxylated type nonionic surfactant is Triton X-100.

[0152] According to the present invention, the reducing agent for the solution of the resuspended dried analyte 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 organophosphorus 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 organophosphorus reducing agent. In a more preferred embodiment, the organophosphorus reducing agent is tricarboxyethylphosphine.

[0153] According to the present invention, the water-miscible polar organic solvent for resuspending the dried analyte in the solution can be of 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 categories 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 every member of the above categories is water-miscible; however, those skilled in the art can readily determine that members of a particular category are water-miscible. In yet more preferred embodiments, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof.

[0154] According to the present invention, the acid in the solution for resuspending the dried analyte can be of 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 an even more preferred embodiment, the carboxylic acid is a monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤4, for example, about ≤3, such as about ≤2, suitably ≤1. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤0.5. In a preferred embodiment, the carboxylic acid is a C1-C10 haloalkyl monocarboxylic acid, a C1-C10 haloalkyl dicarboxylic acid, or a C1-C10 haloalkyl tricarboxylic acid. For example, the carboxylic acid may be a C1-C5 haloalkyl monocarboxylic acid. In an even more preferred embodiment, the carboxylic acid is trifluoroacetic acid (TFA). Therefore, in some preferred embodiments, the acid in the solution for resuspending the dried analyte is TFA.

[0155] All pKa values ​​disclosed in this paper were measured in water at 25°C and 1 atmosphere.

[0156] In a more preferred embodiment, the surfactant is Triton X-100, the reducing agent is tricarboxyethylphosphine, the polar organic solvent is acetonitrile and dimethylformamide, and the acid is TFA.

[0157] In some preferred embodiments, the concentration of acetonitrile in the solution of the dried analyte is between 2% (v / v) and 8% (v / v), more preferably between 3% (v / v) and 7% (v / v), more preferably between 4% (v / v) and 6% (v / v), and even more preferably about 5% (v / v).

[0158] In some preferred embodiments, the concentration of dimethylformamide in the solution of the dried analyte is between 0.1% (v / v) and 3% (v / v), more preferably between 0.5% (v / v) and 2% (v / v), even more preferably between 0.5% (v / v) and 1.5% (v / v), and even more preferably about 1% (v / v).

[0159] In some preferred embodiments, the concentration of trifluoroacetic acid (TFA) in the solution of the dried analyte is between 0.1% (v / v) and 5% (v / v), more preferably between 0.2% (v / v) and 4% (v / v), more preferably between 0.2% (v / v) and 3% (v / v), more preferably between 0.2% (v / v) and 2.5% (v / v), and even more preferably about 0.5% (v / v).

[0160] In some preferred embodiments, the concentration of Triton X-100 in the solution of the dried analyte is between 0.01% (v / v) and 2% (v / v), more preferably between 0.05% (v / v) and 1% (v / v), more preferably between 0.05% (v / v) and 0.8% (v / v), more preferably between 0.05% (v / v) and 0.1% (v / v), and even more preferably about 0.05% (v / v).

[0161] In some preferred embodiments, the concentration of tricarboxyethylphosphine in the solution of the dried analyte is between 0.05% (w / v) and 0.3% (w / v), more preferably between 0.1% (w / v) and 0.2% (w / v), more preferably between 0.12% (w / v) and 0.16% (w / v), and even more preferably about 0.14% (w / v).

[0162] In some preferred embodiments, the solution for dissolving the analyte is an aqueous solution containing a surfactant and a reducing agent. In an even more preferred embodiment, the solution for dissolving the dried analyte contains Triton X-100 at a concentration between 0.05% (v / v) and 0.8% (v / v) and tricarboxyethylphosphine at a concentration between 0.1% (w / v) and 0.2% (w / v).

[0163] In another preferred embodiment, the solution for dissolving the dried analyte comprises Triton X-100 at concentrations between 0.01% (v / v) and 0.1% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), and acetonitrile at concentrations between 3% (v / v) and 7% (v / v).

[0164] In some preferred embodiments, the solution for dissolving the analyte is an aqueous solution comprising a surfactant, a reducing agent, a polar organic solvent, and an acid. In an even more preferred embodiment, the solution for dissolving the dried analyte comprises Triton X-100 at concentrations between 0.01% (v / v) and 0.1% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v). In a more preferred embodiment, the solution dissolving the dried analyte comprises Triton X-100 at concentrations between 0.01% (v / v) and 0.1% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), dimethylformamide at concentrations between 0.1% (v / v) and 3% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v).

[0165] In some embodiments, samples prepared by the method of the present invention are further analyzed by liquid chromatography and / or mass spectrometry.

[0166] The present invention also relates to a method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, characterized in that the method does not involve immunoprecipitation or digestion of the plasma sample prior to mass spectrometry analysis.

[0167] This invention also relates to a method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis, characterized in that the method mainly includes the following steps:

[0168] a) Contact the plasma sample with a denaturing agent.

[0169] b) Perform a first solid-phase extraction on the solution obtained in step a) to recover the first eluent.

[0170] c) Perform a second solid-phase extraction step on the first eluent obtained in step b) to recover the second eluent, and

[0171] d) Dry the second eluent obtained in step c) and process it for mass spectrometry analysis.

[0172] The sample obtained from step d) contains intact amyloid β peptides Aβ40 and Aβ42.

[0173] In a second aspect, the present invention relates to a method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry. In some embodiments, the method of the present invention for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry includes steps a) to d) of the method for preparing a sample as described herein, and further includes the following steps:

[0174] i) Perform liquid chromatography on the solution obtained in step d) to separate the analyte of interest.

[0175] ii) Ionize the analytes separated in step i) to produce one or more charged substances;

[0176] iii) Separate the one or more charged substances based on the ion mobility of the one or more charged substances.

[0177] iv) Detect one or more of the charged substances separated in step iii) and measure their abundance by mass spectrometry; and

[0178] v) Determine the amount or concentration of intact amyloid β-peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged substances measured in step iv) with a standard curve.

[0179] In some embodiments of the invention, the method for quantifying intact amyloid peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry includes performing a chromatographic step i) on a solution obtained from a method as described herein for preparing a sample containing amyloid β peptides. In a preferred embodiment, the chromatographic step is liquid chromatography (LC). In a more preferred embodiment, the liquid chromatography is HPLC. In yet more preferred embodiments, the chromatographic step is micro-HPLC.

[0180] In some embodiments of the invention, the method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry includes a second step ii), wherein the analytes separated in step i) are ionized to produce one or more charged substances.

[0181] In some embodiments of the present invention, the analyte undergoes positive ion mode electrospray ionization (ESI). The conditions for performing the electrospray ionization of the present invention are well known to those skilled in the art.

[0182] In some embodiments of the present invention, the method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry includes a third step iii), wherein one or more charged substances obtained in step ii) are separated according to their ion mobility. In a preferred embodiment, the one or more charged substances 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).

[0183] In some embodiments of the invention, after separating charged substances according to their ion mobility, they are detected by mass spectrometry and their abundance is measured (step iv). As previously explained, the term "mass spectrometry" includes several analytical techniques for determining the mass-to-charge ratio of an analyte or a group of analytes. Non-limiting examples of mass spectrometry techniques include ion traps (3D or linear), single quadrupole or triple quadrupole, time-of-flight, orbital ion traps, Fourier transform ion cyclotron resonance mass spectrometry, and combinations thereof (hybrid instruments).

[0184] In a preferred embodiment, the technique for measuring the intensity and abundance of charged substances by mass spectrometry is multiple reaction monitoring (MRM) in a triple quadrupole apparatus.

[0185] In some embodiments of the present invention, the method for quantifying intact amyloid β peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry includes a fifth step v), wherein the amount or concentration of said peptide is determined by comparing the abundance of the analyte measured in iv) with a standard curve.

[0186] Therefore, calibration curves are preferably prepared using at least one standard at incremental concentrations to quantify the amount or concentration of the analyte in the sample analyzed by mass spectrometry. In the context of this invention, since Aβ40 and Aβ42 are preferably the peptides to be quantified, the labeled... 15 N-Aβ40 and 15 N-Aβ42 is preferably used as a standard for calibration curves.

[0187] Calibration curves can be prepared using buffer solutions containing BSA, such as PBS, as described in the prior art. However, in a preferred embodiment of the invention, the calibration curves are prepared using plasma, which provides several advantages, such as providing equal recoveries for both standards and analytes, and equalizing matrix effects that could otherwise adversely affect quantification. In a more preferred embodiment, the calibration curves are prepared using human plasma.

[0188] Furthermore, internal standards are used as quality controls for different steps in the method of the present invention and as controls for signal normalization. Therefore, to ensure the quality of different steps, internal standards can be added to the solution used to prepare the calibration curve and to the sample to be analyzed by mass spectrometry.

[0189] In some implementations, the solutions and samples used for calibration curves are labeled as internal standards. 2 H-Aβ40 and 2 H-Aβ42 spiked. In other embodiments, the solution and sample used for calibration curves are labeled as internal standards. 13 C-Aβ40 and 13 C-Aβ42 spiked. In other embodiments, the solution and sample used for calibration curves are used as internal standards. 2 H-Aβ40 and 13 C-Aβ42 or 2 H-Aβ42 and 13 C-Aβ40 spiked.

[0190] In other embodiments, the present invention relates to a method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in a plasma sample by mass spectrometry, characterized in that the method does not include the step of determining the concentration of the peptides by comparing the abundance of the analytes measured in iv) with a standard curve. In such embodiments, the method used for quantification is a semi-quantitative method, wherein the purpose of using internal standards is to quantify the abundance of the analytes without comparison with a standard curve.

[0191] The sample containing amyloid β-peptide used in the method of the present invention is preferably a plasma sample. The volume of plasma sample used in step a) of the method for preparing a sample for mass spectrometry analysis can be determined by a technician considering the specific mass spectrometry protocol to be followed.

[0192] However, one advantage of the method of the present invention is that it reduces the sample volume required to obtain accurate values ​​of amyloid-β peptides Aβ40 and Aβ42 concentrations for identifying early stages of neurodegenerative diseases such as Alzheimer's disease in subjects. The reduced volume of plasma sample required in the method of the present invention is particularly convenient for large-scale screening studies.

[0193] Therefore, the sample volume used in step a) of the method for preparing a plasma sample containing amyloid β-peptide for mass spectrometry analysis 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 yet another more preferred embodiment, the sample volume is about 200 μL.

[0194] In a third aspect, the present invention relates to an aqueous solution for treating dried eluent 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 other embodiments, the aqueous solution comprises a surfactant and a reducing agent. In other embodiments, the solution comprises a surfactant, a reducing agent, a polar organic solvent, and an acid.

[0195] According to the present invention, the surfactant used to process the solution of the dried analyte can be of any type known to those skilled in the art, preferably nonionic. In some embodiments, the surfactant is nonionic. In some preferred embodiments, the nonionic surfactant is an ethoxylated type. In yet more preferred embodiments, the ethoxylated type nonionic surfactant is Triton X-100.

[0196] According to the present invention, the reducing agent used to process the solution of the dried analyte 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 organophosphorus 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 organophosphorus reducing agent. In a more preferred embodiment, the organophosphorus reducing agent is tricarboxyethylphosphine.

[0197] According to the present invention, the water-miscible polar organic solvent used to process the dried analyte solution 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 categories 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 every member of the above categories is water-miscible; however, those skilled in the art can readily determine that members of a particular category are water-miscible. In a further preferred embodiment, the water-miscible organic solvent is acetonitrile, dimethylformamide, or a combination thereof.

[0198] According to the present invention, the acid used to process the solution of the dried analyte can be of 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 an even more preferred embodiment, the carboxylic acid is a monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤4, for example, about ≤3, such as about ≤2, suitably ≤1. The monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid may have a pKa of about ≤0.5. In a preferred embodiment, the carboxylic acid is a C1-C10 haloalkyl monocarboxylic acid, a C1-C10 haloalkyl dicarboxylic acid, or a C1-C10 haloalkyl tricarboxylic acid. For example, the carboxylic acid may be a C1-C5 haloalkyl monocarboxylic acid. In an even more preferred embodiment, the carboxylic acid is trifluoroacetic acid (TFA). Therefore, in some preferred embodiments, the acid used to process the solution of the dried analyte is TFA.

[0199] In a more preferred embodiment, the surfactant is Triton X-100, the reducing agent is tricarboxyethylphosphine, the polar organic solvent is acetonitrile and dimethylformamide, and the acid is TFA.

[0200] In a more preferred embodiment, the aqueous solution of the present invention for resuspending the dried eluent to be analyzed by mass spectrometry comprises Triton X-100 at concentrations between 0.01% (v / v) and 0.1% (v / v) and tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v).

[0201] In other preferred embodiments, the aqueous solution of the present invention for treating the dried eluent to be analyzed by mass spectrometry comprises Triton X-100 at concentrations between 0.010% (v / v) and 1% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), dimethylformamide at concentrations between 0.1% (v / v) and 3% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v).

[0202] The invention will be more fully understood by referring to the following examples. However, they should not be construed as limiting the scope of the invention. Example

[0203] Example 1: Preparation of plasma samples for mass spectrometry analysis

[0204] Calibration curves and quality control

[0205] Using human blood plasma and 15 N-Aβ40 and 15Calibration curves and quality control samples were prepared using N-Aβ42-labeled standards (rPeptide, Watkinsville, GA, USA). Calibration ranges of 50 pg / ml to 3000 pg / ml and 10 pg / ml to 100 pg / ml were used to prepare a series of... 15 N-Aβ40 standard and 15 N-Aβ42 standard.

[0206] The calibration curve's series of standards, quality control samples, and plasma samples from human subjects awaiting further analysis were all used as internal standards with custom-labeled labels. 2 H-Aβ40 and 2 H-Aβ42 (Bachem, Bubendorf, Switzerland) is spiked for quality control and signal normalization.

[0207] Preparation of plasma samples for analysis by mass spectrometry

[0208] Plasma samples obtained from human subjects are prepared according to different protocols (A, B, C, or D), depending on the combination of SPE columns subsequently used.

[0209] Protocol A: First, the sample is spiked with the selected internal standard. Then, the sample is denatured by contacting 400 μl of 50% formic acid (FA) in water with 200 μl of human plasma. After denaturation, the amyloid peptide is purified using two consecutive SPE steps.

[0210] The first SPE consists of a reversed-phase SPE using an HLB Prime extraction plate (OASIS HLB Prime 96-well plate, 30 mg, Waters, Milford, MA, USA, part number 186008054). The suitable conditions for the first SPE are outlined below:

[0211] Solventization: 1 ml acetonitrile

[0212] • Adjustment: Add 0.1% FA (formic acid) to 1 ml of water.

[0213] Sample loading

[0214] Wash 1: 0.5ml 5% methanol

[0215] Wash 2: 0.5 ml water with 10% acetonitrile

[0216] Elution: 0.4 ml acetonitrile / water 70 / 30 with 0.1% TFA

[0217] • Acidify the eluent by adding 10 μl of 50% FA to water.

[0218] Following the first SPE, the eluent undergoes a second SPE, in which mixed-mode reversed-phase cation exchange SPE is performed using an MCX extraction plate (OASIS MCX 96-well μElution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below:

[0219] Solventization: 0.2 ml methanol

[0220] • Adjustment: Add 5% FA to 0.2ml of water

[0221] • Sample loading: Acidified eluent from SPE 1

[0222] • Washing 1: 0.4ml water with 5% FA

[0223] Wash 2: 0.4 ml of water with 40% acetonitrile

[0224] • Washing solution 3: 0.4 ml 100% acetonitrile

[0225] • Elution: 100 μl acetonitrile / water / ammonium hydroxide 75 / 15 / 10.

[0226] Following the second SPE, the eluent was evaporated to dryness at 45°C in a vacuum concentrator for 35 minutes. The dried sample was then resuspended in 25 μl of AB solvent, an internally optimized solution containing the following:

[0227] • 100% acetonitrile: 2.5ml

[0228] • 100% Dimethylformamide (DMF): 0.5ml

[0229] · 25% TFA in water: 1 ml

[0230] • 10% Triton X-100 in water: 0.25ml

[0231] • Tricarboxyethylphosphine: 70mg

[0232] • Water: Dilute to 50 ml in a volumetric flask.

[0233] Option B: First, the sample is spiked with the selected internal standard. Then, the sample is denatured by contacting 400 μl of 50% formic acid (FA) in water with 200 μl of human plasma. After denaturation, the amyloid peptide is purified using two consecutive SPE steps.

[0234] The first SPE consists of a reversed-phase SPE using an HLB extraction plate (OASIS HLB, 96-well plate, 30 μm, 30 mg, Waters, Milford, MA, USA, part number WAT058951). The suitable conditions for the first SPE are outlined below:

[0235] Solventization: 1 ml acetonitrile

[0236] • Adjustment: Add 0.1% TFA (trifluoroacetic acid) to 1 ml of water.

[0237] Sample loading

[0238] Wash 1: 1ml 0.1% TFA

[0239] Wash 2: 0.5 ml water with 10% acetonitrile

[0240] • Elution: 0.7 ml acetonitrile / water 30 / 70 2% Triton X-100

[0241] • Acidify the eluent by adding 10 μl of 50% FA to water.

[0242] Following the first SPE, the eluent undergoes a second SPE, in which mixed-mode reversed-phase cation exchange SPE is performed using an MCX extraction plate (OASIS MCX 96-well μElution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below:

[0243] Solventization: 0.2 ml methanol

[0244] • Adjustment: Add 25% FA to 0.2ml of water

[0245] • Sample loading: Acidified eluent from SPE 1

[0246] • Washing 1: 0.4ml water with 25% FA

[0247] Wash 2: 0.4 ml of water with 60% acetonitrile

[0248] • Washing solution 3: 0.4 ml 100% acetonitrile

[0249] • Elution: 100 μl acetonitrile / water / ammonium hydroxide 75 / 15 / 10.

[0250] Following the second SPE, the eluent was evaporated to dryness at 45°C in a vacuum concentrator for 35 minutes. The dried sample was then resuspended in 25 μl of AB solvent, an internally optimized solution containing: 5% v / v AcN in water, 1% v / v dimethylformamide, 0.5% v / v TFA, 0.05% v / v Triton X-100, and 0.14% w / v tricarboxyethylphosphine.

[0251] Option C: First, the sample is spiked with the selected internal standard. Then, the sample is denatured by contacting 400 μl of 50% formic acid (FA) in water with 200 μl of human plasma. After denaturation, the amyloid peptide is purified using two consecutive SPE steps.

[0252] The first SPE consisted of a mixed-mode reversed-phase cation exchange SPE, which was performed using an MCX extraction plate (OASIS MCX 96-well plate, 30 μm, 30 mg, Waters, Milford, MA, USA, part number 186000248). The suitable conditions for the first SPE are outlined below:

[0253] Solventization: 1 ml methanol

[0254] • Adjustment: 25% FA in 1ml of water

[0255] Sample loading

[0256] Washing 1: 1 ml of water with 25% FA

[0257] Wash 2: 1 ml water with 60% acetonitrile (40 / 60)

[0258] • Washing solution 3: 1 ml 100% acetonitrile

[0259] • Elution: 2 × 400 μl acetonitrile / water / ammonium hydroxide (75 / 15 / 10).

[0260] Following the first SPE, the eluent undergoes a second SPE, in which mixed-mode reversed-phase anion exchange SPE is performed using MAXμElution extraction plates (OASIS MAXμElution plates, Waters, Milford, MA, USA, part number 186001829). Suitable conditions for the second SPE are outlined below:

[0261] • Solventization: 0.3 ml methanol

[0262] Adjustment: 0.4ml NH4OH 10%

[0263] Sample loading

[0264] Washing 1: 0.4ml NH4OH 10%

[0265] Wash 2: 0.4ml acetonitrile / water (60 / 40)

[0266] • Elution: 2 x 50 μl acetonitrile / water 70 / 30, 5% TFA.

[0267] Following the second SPE, the eluent was evaporated to dryness at 45°C in a vacuum concentrator for 35 minutes. The dried sample was then resuspended in 25 μl of AB solvent, an internally optimized solution containing: 5% v / v AcN in water, 1% v / v dimethylformamide, 0.5% v / v TFA, 0.05% v / v Triton X-100, and 0.14% w / v tricarboxyethylphosphine.

[0268] Protocol D: First, the sample is spiked with the selected internal standard. Then, the sample is denatured by contacting 200 μl of human plasma with 25% NH4OH in 400 μl of water. After denaturation, the amyloid peptide is purified using two consecutive SPE steps.

[0269] The first SPE consisted of a mixed-mode reversed-phase anion exchange SPE using an OASIS MAX 96-well plate (30 μm, 30 mg, Waters, Milford, MA, USA, part number 186000373). The suitable conditions for the first SPE are outlined below:

[0270] Solventization: 1 ml methanol

[0271] Adjustment: 1 ml NH4OH 10%

[0272] Sample loading

[0273] Washing 1: 1 ml 10% NH4OH

[0274] Wash 2: 1ml acetonitrile / water 60 / 40

[0275] Elution: 2×400μl acetonitrile / water 70 / 30, 5% trifluoroacetic acid.

[0276] Following the first SPE, the eluent undergoes a second SPE, in which mixed-mode reversed-phase cation exchange SPE is performed using an MCX extraction plate (OASIS MCX 96-well μElution plate, 30 μm, 2 mg, Waters, Milford, MA, USA, part number 186001830BA). Suitable conditions for the second SPE are outlined below:

[0277] Solventization: 0.2 ml methanol

[0278] • Adjustment: Add 25% FA to 0.2ml of water

[0279] Sample loading

[0280] • Washing 1: 0.4ml water with 25% FA

[0281] Wash 2: 0.4 ml of water with 60% acetonitrile

[0282] • Washing solution 3: 0.4 ml 100% acetonitrile

[0283] • Elution: 100 μl acetonitrile / water / ammonium hydroxide 75 / 15 / 10.

[0284] Following the second SPE, the eluent was evaporated to dryness at 45°C in a vacuum concentrator for 35 minutes. The dried sample was then resuspended in 25 μl of AB solvent, an internally optimized solution containing the following:

[0285] • 100% acetonitrile: 2.5ml

[0286] • 100% Dimethylformamide (DMF): 0.5ml

[0287] · 25% TFA in water: 1 ml

[0288] • 10% Triton X-100 in water: 0.25ml

[0289] • Tricarboxyethylphosphine: 70mg

[0290] • Water: Dilute to 50 ml in a volumetric flask.

[0291] In each case, the calibration curves and quality control samples also undergo the same preparation process.

[0292] Example 2: Mass spectrometry analysis of plasma samples

[0293] The plasma sample prepared in Example 1 was then analyzed by mass spectrometry according to the amyloid peptide quantification method of the present invention.

[0294] The analytical system for quantifying amyloid peptides includes the following modules:

[0295] oM3 Micro-HPLC dual-pump (trap-elution) chromatograph, equipped with CTC autosampler and column oven (Sciex, Framingham, MA, USA).

[0296] The o6500+QTRAP hybrid triple quadrupole-linear ion trap mass spectrometer is coupled with the SelexION differential mobility spectroscopy-DMS interface (Sciex, Framingham, MA, USA).

[0297] After injection, the sample was loaded onto the trapping column (YMC Triart C18, 12nm, 3μm, 5x0.3mm, YMC, Dinslaken, Germany). Using 0.5% TFA and 5% dimethyl sulfoxide (DMSO) in water, the trapping flow rate was 50 μl / min for 2 minutes. The trapping valve was then switched, and the analyte was eluted from the trapping column to the analytical column and MS system. The following chromatographic conditions were used:

[0298] oA stage: 0.1% FA in water

[0299] oB stage: 0.1% FA in acetonitrile

[0300] Flow rate: 15 μl / min

[0301] o-column: HALO Protein C18, 3.4μm, 0.3x50mm (Advanced Materials Technology, Wilmington, DE, USA)

[0302] Column temperature: 55℃

[0303] Gradient: 15% B for 0.3 min, linearly rises to 40% B in 3.5 min, rises to 90% B in 0.1 min and lasts for 0.3 min, return to the initial condition (15% B) in 0.1 min and lasts for 3 min to allow the column to rebalance.

[0304] At 5.5 min, switch the analysis valve and wash the system with 0.1% Triton X-100 in TFE / water 80 / 20. Then equilibrate the trap column with the loading solvent (5% DMSO and 0.5% TFA in water).

[0305] Sample acquisition begins when the trapping valve is switched and the analyte is eluted onto the analytical column. After chromatographic separation, the analyte enters the ion source of the mass spectrometer and undergoes electrospray ionization (ESI) in positive ion mode. Upon entering the gas phase, the analyte undergoes differential mobility spectrometry (DMS) to separate it from interfering matrix ions as much as possible.

[0306] The specific combination of DMS and microLC results in reduced background noise and improved sensitivity.

[0307] After separation by DMS, the ions (charged substances of the analyte of interest) are analyzed by multiple reaction monitoring (MRM) in a triple quadrupole analyzer. In short, the precursor (pseudomolecule) ions are filtered in the first quadrupole (Q1), fragmented in the second quadrupole (Q2 or collision cell) by collisions with the target gas (a process called collision-induced dissociation, CID, or collision-activated dissociation, CAD), and the fragment of interest is filtered in the third quadrupole (Q3) and reaches the detector.

[0308] Precursor ions with charge state 5 (z=5) were selected in Q1, fragmented in Q2 by collision with nitrogen, and the fragments were analyzed in Q3 (also z=5). The MRM acquisition parameters are summarized below:

[0309]

[0310] For Aβ40, 15 N-Aβ40 and 2 H-Aβ40, b was analyzed in Q3 39 5+ Product ions. For Aβ42, 15 N-Aβ42 and 2 H-Aβ42, b was analyzed in Q3 41 5+ Product ions. All molecular masses in the table above are average masses.

[0311] The following acquisition parameters were consistent for all substances: source temperature 250°C, curtain gas 30 psi, ion spray voltage 4800 V, ion source gas 1 (nebulization) 30 psi, ion source gas 2 (desolvation) 50 psi, declustering potential 85 V, and inlet potential 10 V. Both filter quadrupoles, Q1 and Q3, operated at unit resolution.

[0312] By linear regression of the calibration curve samples, the peak area ratio was fitted with a linear equation. 15 N-Aβ40 / 2 H-Aβ40 and 15 N-Aβ42 / 2 H-Aβ42) and concentration data. In the case of quality control samples, the response ratio ( 15 N-Aβ40 / 2 H-Aβ40 and 15 N-Aβ42 / 2 H-Aβ42) was interpolated in its corresponding calibration curve to obtain the calculated concentration for evaluation. For the sample of interest, the sample response ratio (Aβ40 / 2 H-Aβ40 and Aβ42 / 2H-Aβ42) was interpolated within its corresponding calibration curve to obtain the calculated concentration. Regression analysis was performed using MultiQuant 3.0.3 software (Sciex, Framingham, MA, USA).

[0313] Example 3: Quantification of intact amyloid β-peptides Aβ40 and Aβ42 in plasma samples prepared and analyzed according to the present invention

[0314] Twenty replicates from two different plasma samples (sample A and sample B) were prepared according to the methods described in Examples 1 (Scheme B) and 2, and further analyzed by mass spectrometry according to the method explained in Example 2.

[0315] The values ​​obtained by interpolation in the calibration curves for 20 replicates of amyloid β-peptide quantification for each plasma sample are shown in Table 1 below.

[0316] As can be observed in Table 1, the quantified coefficient of variation (%CV) for Aβ40 is less than 4%, while the quantified coefficient of variation for Aβ42 is approximately 6%. These coefficients of variation represent small variability, which makes the method of the present invention suitable for detecting small differences in Aβ peptides between groups.

[0317] Table 1. Quantification of Aβ40 and Aβ42 in plasma

[0318]

[0319] The absolute values ​​of Aβ40 quantified in human plasma are similar to those obtained by MS methods and immunoligand binding assays (LBA) methods (primarily ELISA) known in the art. However, for the quantification of Aβ42, the levels observed using the method of this invention are significantly higher.

[0320] Furthermore, the method of the present invention allows for the quantification of intact peptides Aβ40 and Aβ42, while other methods known in the art, such as those of the Bateman group, detect truncated peptides (Ovod et al., Amyloid B concentrations and stable isotope labelling kinetics of human plasma specific to central nervous system amyloidosis, Alzheimer's and Dementia, 2017 Oct; 13(10):1185).

[0321] Example 4: Detection of small changes in Aβ substance in a sample

[0322] In further research, the ability to detect small differences in amyloid protein concentration in human plasma was investigated. Therefore, 200 μl and 220 μl plasma samples were prepared according to the method of Example 1 (Protocol B) and further analyzed by mass spectrometry according to the method explained in Example 2. In this case, the ratio (area) of the analyte was... 14 N-Aβ40 / 15 N-Aβ40 and 14 N-Aβ42 / 15 Compare with N-Aβ42).

[0323] In this embodiment, the amyloid concentration in the plasma sample was exactly the same. However, this is not the case in absolute terms, as there is a 10% difference in volume. This situation simulates a 10% concentration difference in real samples.

[0324] Table 2 shows the results of Aβ42 and Aβ40 quantifications performed in several replicates of the same plasma samples with an initial sample volume of 200 μl or 220 μl, according to the method of the present invention as described in Examples 1 and 2.

[0325] The results showed that, quantified by the method of the present invention, a 10% increase in plasma sample volume resulted in a 10% and 9% increase in the amounts of Aβ42 and Aβ40, respectively.

[0326] Table 2. Determination of a 10% increase in Aβ40 and Aβ42 peptides in plasma samples

[0327]

[0328] Example 5: Diagnostic performance of the method of the present invention

[0329] Samples from 36 individuals (26 PET-negative and 14 PET-positive individuals) previously characterized by positron emission tomography (PET) were prepared according to the method explained in Example 1, and further analyzed by mass spectrometry according to the method explained in Example 2. The Aβ42 / Aβ40 ratio was calculated, and... Figure 1 The results show that for PET-negative individuals, the mean Aβ42 / Aβ40 ratio was 0.184 with a standard deviation of 0.02; while for PET-positive individuals, the mean Aβ42 / Aβ40 ratio was 0.152 with a standard deviation of 0.03 (p < 0.001). The ROC curve shows AUC = 0.8365 (…). Figure 2 ).

[0330] As explained in the background section, the Aβ42 / Aβ40 concentration ratio has been used as a biomarker for cerebral amyloidosis in the early stages of Alzheimer's disease, where Aβ42 concentrations are lower when amyloid plaques are present. Therefore, these results indicate that the methods of the present invention can be used not only to monitor changes in Aβ as a response to treatment but also for diagnostic purposes, as they allow for differentiation between PET-positive and PET-negative individuals.

[0331] Example 6: Comparison of different combinations of SPE columns

[0332] Based on the preparation of samples for mass spectrometry as explained in Example 1 and then the analysis as described in Example 2, different tests were performed on the SPE column combination. The chromatographic traces extracted with Aβ40 and Aβ42, respectively, were... Figures 4 to 8 As shown in the figure. For comparison, the use of a single SPE step (mixed-mode reversed-phase cation exchange) was also evaluated. Figure 3 Due to the duration of column chromatography, the resulting eluents are not easily analyzed successfully by Micro-LC; therefore, the use of single reverse mode SPE was not evaluated.

[0333] Therefore, the following SPE combinations were evaluated:

[0334] - Figure 4 The chromatographic traces for the extraction of Aβ40 (left panel) and Aβ42 (right panel) are shown when plasma samples are contacted with an acidic denaturant according to scheme A and separated first by reverse mode SPE (HLB Prime) and then by a second mixed mode reversed-phase cation exchange SPE (MCX).

[0335] - Figure 6 The chromatographic traces for the extraction of Aβ40 (left panel) and Aβ42 (right panel) are shown when plasma samples are contacted with an acidic denaturant according to scheme B and separated first with reversed-mode SPE (HLB) and then with a second mixed-mode reversed-phase cation exchange SPE (MCX).

[0336] - Figure 5 The chromatographic traces for Aβ40 (left panel) and Aβ42 (right panel) extractions are shown when plasma samples are contacted with an acidic denaturing agent and separated first using a mixed-mode reversed-phase cation exchange SPE (MCX) followed by a second reversed-phase SPE (HLB).

[0337] The figures clearly show that when the first SPE is MCX followed by HLB, the traces extracted for Aβ40 (left panel) and Aβ42 (right panel) are worse than those obtained using either the first HLB or HLB Prime followed by MCX. Furthermore, when compared to a single SPE step, as is known in the prior art... Figure 3 The trace is much stronger, which suggests that the combination of two consecutive SPE steps provides a better signal-to-noise ratio and therefore better sensitivity.

[0338] - Figure 8 The chromatographic traces for Aβ40 (left panel) and Aβ42 (right panel) extraction are shown when a plasma sample is contacted with an acidic denaturing agent and separated first using a mixed-mode reversed-phase cation exchange SPE (MCX) followed by a second mixed-mode reversed-phase anion exchange SPE (MAX) (Scheme C). These traces are compared with those obtained when the plasma sample, denatured with an acidic agent, is first separated using a reversed-mode SPE (HLB) followed by a second mixed-mode reversed-phase cation exchange SPE (MCX).

[0339] Figure 8 The chromatographic traces shown indicate that using the first MCX SPE followed by the second MAX SPE allows for the acquisition of identifying peaks for Aβ40 and Aβ42, similar to using the first HLB SPE followed by the MCX SPE, although less intense. However, when the first SPE was replaced by HLB, the recoveries of both analytes were very low.

[0340] - Figure 7 The chromatographic traces for Aβ40 (left panel) and Aβ42 (right panel) extractions are shown when plasma samples are contacted with a basic denaturing agent according to scheme D, and separation is performed first with a mixed-mode reversed-phase anion exchange SPE (MAX) followed by a second mixed-mode reversed-phase cation exchange SPE (MCX). For comparison, in Figure 7 The diagram also shows the traces obtained when plasma samples are denatured with an alkaline agent and separated first by reversed-phase SPE (HLB) followed by a second mixed-mode reversed-phase cation exchange SPE (MCX).

[0341] These results indicate that when plasma samples are denatured with an alkaline agent and then undergo a first MAX followed by an MCX, the peaks obtained for Aβ40 and Aβ42 are almost as good as the combination of HLB followed by an MCX when the sample is denatured with an acidic agent.

Claims

1. Process for the preparation of a plasma sample comprising amyloid β peptide for analysis by mass spectrometry, characterized in that The method includes the following steps: a) Contact the plasma sample with a denaturing agent; b) Perform a first solid-phase extraction on the solution obtained in step a) to recover the first eluent. c) Perform a second solid-phase extraction step on the first eluent obtained in step b) to recover the second eluent, and d) Dry the second eluent obtained in step c) and process it for mass spectrometry analysis. The sample obtained from step d) contains intact amyloid β peptides Aβ40 and Aβ42. - Wherein the first solid-phase extraction step is reversed-phase solid-phase extraction, and the second solid-phase extraction step is cation exchange solid-phase extraction. - Wherein the first solid-phase extraction step is anion-exchange solid-phase extraction, and the second solid-phase extraction step is cation-exchange solid-phase extraction, or - Wherein the first solid-phase extraction step is cation exchange solid-phase extraction, and the second solid-phase extraction step is anion exchange solid-phase extraction. and - Wherein, if the first solid-phase extraction step is reversed-phase solid-phase extraction or cation exchange solid-phase extraction, then the denaturing agent is an acidic denaturing agent, or - Wherein, if the first solid-phase extraction step is anion exchange solid-phase extraction, the denaturant is a basic denaturant.

2. The method according to claim 1, wherein the cation exchange solid-phase extraction is a strong, weak, or mixed mode of reversed-phase cation exchange.

3. The method according to claim 1, wherein the anion exchange solid-phase extraction is a strong, weak, or mixed mode of reverse anion exchange.

4. The method according to any one of claims 1 to 3, wherein in step a), the plasma sample is contacted with an acidic denaturing agent to obtain a solution having a pH of less than or equal to about 4.

5.

5. The method according to claim 4, wherein the acidic denaturant is a solution of formic acid in water with a concentration between 40% (v / v) and 70% (v / v).

6. The method according to any one of claims 1 to 3, wherein each of the first solid phase extraction step and the second solid phase extraction step comprises at least two washing steps, characterized in that, The first solid-phase extraction step and the first washing step of the second solid-phase extraction step are performed with a solution containing acid, and the second washing step of the first solid-phase extraction step and the second solid-phase extraction step are performed with a solution containing a water-miscible polar organic solvent.

7. The method of claim 6, wherein the acid-containing solution in the first washing step of the first solid-phase extraction step is the same as the acid-containing solution in the first washing step of the second solid-phase extraction step.

8. The method of claim 6, wherein the acid-containing solution in the first washing step of the first solid-phase extraction step is different from the acid-containing solution in the first washing step of the second solid-phase extraction step.

9. The method according to any one of claims 1 to 3, wherein each of the first solid phase extraction step and the second solid phase extraction step comprises at least two washing steps, characterized in that, The first washing step of the first solid-phase extraction is performed with a solution containing an acid, and the first washing step of the second solid-phase extraction is performed with a solution containing an alkali, and the second washing steps of the first solid-phase extraction and the second solid-phase extraction are performed with a solution containing a polar organic solvent that is miscible with water.

10. The method according to any one of claims 1 to 3, wherein in step a), the plasma sample is contacted with an alkaline denaturant to obtain a solution having a pH greater than or equal to 11.

11. The method of claim 10, wherein the alkaline denaturant is a solution of ammonium hydroxide in water with a concentration between 5% (v / v) and 50% (v / v).

12. The method according to any one of claims 1 to 3, wherein each of the first solid-phase extraction step and the second solid-phase extraction step comprises at least two washing steps, characterized in that, The first washing step of the first solid-phase extraction is performed with a solution containing an alkali, and the first washing step of the second solid-phase extraction is performed with a solution containing an acid, and the second washing steps of the first solid-phase extraction and the second solid-phase extraction are performed with a solution containing a polar organic solvent that is miscible with water.

13. The method according to any one of claims 1-3, 5, 7-8 and 11, wherein the solution for treating the dried eluent in step d) is an aqueous solution containing a surfactant and a reducing agent.

14. The method of claim 13, wherein the solution for treating the dried eluent in step d) is an aqueous solution containing Triton X-100 at concentrations between 0.01% (v / v) and 0.8% (v / v) and tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v).

15. The method according to any one of claims 1-3, 5, 7-8, 11 and 14, wherein the solution for treating the dried eluent is an aqueous solution comprising a surfactant, a reducing agent, a water-miscible polar organic solvent and an acid.

16. The method of claim 15, wherein the solution for treating the dried eluent in step d) is an aqueous solution comprising Triton X-100 at concentrations between 0.01% (v / v) and 0.8% (v / v), tricarboxyethylphosphine at concentrations between 0.1% (w / v) and 0.2% (w / v), acetonitrile at concentrations between 3% (v / v) and 7% (v / v), dimethylformamide at concentrations between 0.1% (v / v) and 3% (v / v), and trifluoroacetic acid (TFA) at concentrations between 0.1% (v / v) and 3% (v / v).

17. The method according to any one of claims 1-3, 5, 7-8, 11, 14 and 16, wherein the plasma sample is a human plasma sample.

18. The method according to any one of claims 1-3, 5, 7-8, 11, 14 and 16, wherein the volume of the plasma sample used in step a) is between 100 μL and 400 μL.

19. The method according to any one of claims 1-3, 5, 7-8, 11, 14, and 16, characterized in that, The method does not include immunoprecipitation or digestion of the plasma sample prior to mass spectrometry analysis.

20. The method according to any one of claims 1-3, 5, 7-8, 11, 14 and 16, characterized in that, The treatment of the eluent in step d) of claim 1 makes the eluent suitable for use in liquid chromatography coupled with mass spectrometry.

21. A method for quantifying intact amyloid β-peptides Aβ40 and Aβ42 in plasma samples by mass spectrometry, characterized in that... The method includes steps a) to d) of the method for preparing a plasma sample according to any one of the preceding claims, and further includes the following steps: i) Perform liquid chromatography on the sample obtained in step d) to separate the analytes of interest. ii) Ionize the analytes separated in step i) to produce one or more charged substances; iii) Separate the one or more charged substances based on the ion mobility of the one or more charged substances. iv) Detect one or more of the charged substances separated in step iii) and measure their abundance by mass spectrometry; and v) Determine the amount or concentration of intact amyloid β-peptides Aβ40 and / or Aβ42 in the plasma sample by comparing the abundance of one or more charged substances measured in step iv) with a standard curve.

22. The method of claim 21, wherein the liquid chromatography is micro-HPLC, the ionization is electrospray ionization (ESI), the separation of the one or more charged substances 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 substances is multiple reaction monitoring (MRM) in a triple quadrupole.

23. The method according to any one of claims 21 or 22, wherein the standard curve is prepared using human plasma.

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