A mass spectrometry method for determining the beta-amyloid 42 / 40 ratio in human plasma

Abstract

This invention provides a mass spectrometry method for determining the 42 / 40 ratio of β-amyloid protein in human plasma, including using¹ 5 Using intact Aβ42 and Aβ40 proteins labeled with N stable isotopes as internal standards, plasma samples were extracted and purified using immunoprecipitation. Characteristic peptides were then generated through Lys-N proteolysis, and finally, targeted quantitative analysis was performed using liquid chromatography-tandem mass spectrometry. The characteristic peptide of Aβ42 is Aβ28-42 KGAIIGLMVGGVVIA; the characteristic peptide of Aβ40 is Aβ28-40 KGAIIGLMVGGVV. Compared to intact proteins, the characteristic peptides in this invention are less prone to aggregation and exhibit higher ionization efficiency in mass spectrometry, demonstrating excellent specificity, repeatability, and sensitivity, providing a reliable basis for the diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry detection technology, and more specifically, to a mass spectrometry method for determining the 42 / 40 ratio of Aβ protein in human plasma. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for 60% to 80% of dementia cases worldwide. With the increasing aging of the population, the burden of AD is becoming increasingly severe: the 2020 Chinese epidemiological survey showed that there were approximately 38.77 million people aged 60 and above with mild cognitive impairment (MCI) and approximately 15.07 million with dementia, of which 9.83 million were AD patients, posing a major public health challenge (see: Chinese Expert Consensus on the Diagnosis and Treatment of Mild Cognitive Impairment of Alzheimer's Disease 2024 [J]).

[0003] Currently, positron emission tomography (PET) of amyloid protein and the β-amyloid 42 / 40 (Aβ42 / 40) ratio and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) are the core methods for pathological diagnosis of Alzheimer's disease (AD) (see: Revised criteria for diagnosis and staging of Alzheimer's disease: Alzheimer's Association Workgroup[J]. Alzheimer's & Dementia, 2024: alz.13859.). However, amyloid PET testing is expensive and carries the risk of radiation exposure, limiting its accessibility to primary healthcare; CSF testing requires invasive procedures such as lumbar puncture, significantly restricting its clinical application. In contrast, blood-based biomarkers, such as the plasma Aβ42 / 40 ratio, have become the focus of research as an alternative due to their convenience, low cost, and high safety. Multiple studies have confirmed that plasma Aβ42 / 40 results are highly consistent with amyloid PET results, suggesting its potential in clinical diagnosis and efficacy assessment.

[0004] However, the clinical application of the plasma Aβ42 / 40 ratio still faces multiple challenges: First, compared to a decrease of about 50% in cerebrospinal fluid (CSF) Aβ42 / 40 levels, individuals with positive brain Aβ pathology show a decrease of less than 20% in plasma Aβ42 / 40 levels; second, peripheral metabolic interference (such as liver clearance and plasma protein binding) may affect the specificity of the Aβ42 / 40 ratio; and third, although current immunoassay techniques are highly automated, they are heavily reliant on antibodies, and their accuracy is generally limited.

[0005] Mass spectrometry (MS / MS) is a more accurate and less specific method for detecting Aβ protein in plasma compared to immunoassay. Several existing technologies utilize MS / MS for this purpose, such as: Patent CN108291916B from Quest Diagnostics Investment Co., Ltd., which discloses a method for detecting amyloid protein β using MS / MS, involving Lys-C cleavage to obtain characteristic peptides; and Patent CN117471007B from Guangdong Provincial Hospital of Traditional Chinese Medicine, which discloses a method for detecting Aβ protein in plasma, primarily utilizing Trypsin cleavage to obtain characteristic peptides. The characteristic peptides obtained from these two patents are identical, including the characteristic peptide GAIIGLMVGGVVIA (SEQ ID NO. 5) for Aβ29–42 and the characteristic peptide GAIIGLMVGGVV (SEQ ID NO. 6) for Aβ29–40. These two characteristic peptides exhibit strong hydrophobicity and low isoelectric points, potentially affecting their stability and ionization efficiency. Because the sensitivity and resolution of conventional ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) platforms are far lower than those of nanoliter liquid chromatography-high resolution mass spectrometry (NHPLC-MS / MS) platforms, it is generally believed that conventional UHPLC-MS / MS platforms are insufficient to meet the β-resolved requirements. There is a need for clinical testing of amyloid protein. The nanoliter liquid chromatography-high resolution mass spectrometry platform used in patent CN117471007B is expensive and usually used in research, which raises questions about its accessibility to clinical applications.

[0006] The patent application CN117990819A of Suzhou WuXi AppTec Biotechnology Co., Ltd. discloses a liquid chromatography-tandem mass spectrometry method for detecting Aβ protein in plasma. It mainly utilizes BrCN chemical specific cleavage of Aβ protein to obtain characteristic peptides, including the characteristic peptide VGGVVIA (SEQ ID NO.7) of Aβ36–42 and the characteristic peptide VGGVV (SEQ ID NO.8) of Aβ36–40. These two characteristic peptides also have the problems of strong hydrophobicity and low isoelectric point. On the other hand, cyanogen bromide is a highly toxic and volatile hazardous chemical, which is very unfriendly to operators.

[0007] Quest Diagnostics Investment Co., Ltd.'s patent application CN114072678A discloses a multiplex assay for determining the 42 / 40 ratio of Aβ protein in human plasma samples. This patent primarily utilizes an immunoassay method, which involves multiple antibodies. This patent application employs an immunoassay method, relying on antibody recognition capabilities, which makes it prone to cross-reactivity with structurally similar Aβ subtypes, leading to insufficient specificity and a high risk of false positives.

[0008] In summary, existing detection methods related to Aβ protein still have the following shortcomings:

[0009] (1) Inadequate selection of characteristic peptides: Most existing methods obtain peptides with strong hydrophobicity and low ionization efficiency, which affect detection sensitivity and stability.

[0010] (2) The method is not suitable for clinical promotion: it either relies on high-end scientific research equipment, uses dangerous reagents, or lacks specificity.

[0011] (3) Lack of LC-MS / MS platform methods suitable for routine medical environments: There are few reports of mature methods based on the clinically popular platform of ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS). Summary of the Invention

[0012] To address the aforementioned technical problems, the present invention aims to provide a mass spectrometry method for determining the 42 / 40 ratio of Aβ protein in human plasma, comprising employing¹ 5 Using N-stable isotope-labeled intact Aβ42 and Aβ40 proteins as internal standards, plasma samples were extracted and purified using immunoprecipitation. Characteristic peptides were then generated through Lys-N proteolysis, and finally, targeted quantitative analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The characteristic peptide of Aβ42 was Aβ28-42KGAIIGLMVGGVVIA (SEQ ID NO.4), with a quantitative ion concentration of 598.4++([b12]²). + The characteristic peptide of Aβ40 is Aβ28-40 KGAIIGLMVGGVV (SEQ ID NO.2), and its quantitative ion is 548.8++ ([b12]²). + The characteristic peptides of this invention can effectively avoid cross-interference from numerous structurally similar subtypes of Aβ40 and Aβ42 in plasma. Compared with intact proteins, the characteristic peptides are less prone to aggregation and have higher ionization efficiency in mass spectrometry. Furthermore, the mass spectrometry precursor ions and mass spectrometry daughter ions of the characteristic peptides have stronger response signals and better stability, resulting in better accuracy, repeatability, and sensitivity of the plasma Aβ42 / 40 ratio detection method. By accurately measuring the Aβ42 / Aβ40 ratio, a reliable basis for the diagnosis of Alzheimer's disease (AD) can be provided.

[0013] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a mass spectrometry method for determining the 42 / 40 ratio of Aβ protein in human plasma, comprising the following steps: S1, Take Resistance β Amyloid antibody is coupled with magnetic beads to obtain magnetic beads coated with Aβ protein antibody; S2. Add a mixed internal standard solution of Aβ40 and Aβ42 to a human plasma sample, then add magnetic beads coated with the Aβ protein antibody and mix and incubate to perform immunologic capture of Aβ protein and internal standard Aβ protein in the human plasma sample to obtain Aβ protein-magnetic bead complex. S3. The Aβ protein on the washed Aβ protein-magnetic bead complex is directly enzymatically digested using Lys-N metalloproteinase, so that the captured natural Aβ protein and the internal standard Aβ protein are respectively enzymatically digested to produce corresponding characteristic peptides, wherein the characteristic peptide used to quantify Aβ42 is KGAIIGLMVGGVVIA (SEQ ID NO.4), and the characteristic peptide used to quantify Aβ40 is KGAIIGLMVGGVV (SEQ ID NO.2). S4. Analyze the enzymatic digestion products by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and simultaneously detect the characteristic peptide signals generated by the natural Aβ protein and the isotopically labeled characteristic peptide signals generated by the internal standard Aβ protein; based on the ratio of the natural characteristic peptide signals to their corresponding isotopically labeled characteristic peptide signals, calculate the concentrations of Aβ42 and Aβ40 in the human plasma sample, and thus obtain the ratio of Aβ42 to Aβ40.

[0014] As some specific embodiments of the present invention, step S1 specifically includes: first resuspending the magnetic beads with phosphate buffer, adding anti-Aβ antibody, mixing thoroughly, and then adding ammonium sulfate solution for incubation.

[0015] As some specific embodiments of the present invention, the incubation temperature is 30~40 ℃, preferably 37 ℃, and the time is 16-24 h.

[0016] As some specific embodiments of the present invention, in step S2, the internal standard of Aβ40 is... 15 The internal standards for N-Aβ40 and Aβ42 are 15 N-Aβ42; in the mixed internal standard solution 15 The final concentration of N-Aβ40 is 50~400 pg / mL, preferably 200 pg / mL. 15 The final concentration of N-Aβ42 is 25~400 pg / mL, preferably 200 pg / mL.

[0017] As some specific embodiments of the present invention, in step S2, the mixed incubation system further includes a mixture of protease inhibitors; and a detergent selected from at least one of CHAPS, Tween20, Igepal CA630, CHAPSO, SDS, and Triton X-100.

[0018] As some specific embodiments of the present invention, in step S2, the temperature of the mixed incubation is 3~10 ℃ and the time is 1~8 h; preferably 4 ℃ for 2 h.

[0019] As some specific embodiments of the present invention, in step S3, the washing includes resuspending and washing the Aβ protein-magnetic bead complex with PBS 2 to 5 times, and then resuspending and washing with ammonium bicarbonate solution.

[0020] As some specific embodiments of the present invention, step S3 specifically includes: adding ammonium bicarbonate solution to the Aβ protein-magnetic bead complex, then adding Lys-N metalloproteinase solution, mixing and incubating with shaking for digestion; after digestion, adding formic acid to terminate the enzymatic reaction, centrifuging the reaction product and collecting the supernatant for subsequent LC-MS / MS analysis. The present invention performs enzymatic digestion directly on magnetic beads, eliminating the traditional steps of elution, lyophilization, and reconstitution, reducing product loss and increasing analytical throughput.

[0021] As some specific embodiments of the present invention, the concentration of the ammonium bicarbonate solution is 25~100 mM, preferably 50 mM; And / or, the concentration of the Lys-N metalloproteinase solution is 1~10 ng / μL, preferably 2 ng / μL; And / or, the temperature of the oscillation incubation is 25~45℃, preferably 37℃, the rotation speed is 1000~1400 rpm, preferably 1400 rpm, and the time is 2~8 h, preferably 4 h; And / or, the centrifugation speed is 12000~16000 rpm, preferably 14000 rpm, and the time is 5~15 min, preferably 10 min.

[0022] As some specific embodiments of the present invention, in step S3, the quantitative ion of the characteristic peptide of Aβ40 is 548.8++; the quantitative ion of the characteristic peptide of Aβ42 is 598.4++.

[0023] As some specific embodiments of the present invention, in step S4, when performing liquid chromatography-tandem mass spectrometry analysis, the chromatographic column is a reversed-phase chromatographic column, and the column temperature is 40~60℃, preferably 50℃. Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is an acetonitrile solution of 0.1% formic acid.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) Advanced detection principle with high specificity and accuracy: This invention uses mass spectrometry for detection, directly performing qualitative and quantitative analysis based on the mass-to-charge ratio of characteristic peptides. This fundamentally avoids the false positive or false negative problems caused by antibody cross-reaction in immunological methods. Compared to existing immunoassay techniques, this method exhibits higher specificity, sensitivity, and accuracy in low-concentration samples, making it particularly suitable for early Alzheimer's disease screening scenarios where the Aβ42 / 40 ratio in plasma shows only a slight decrease.

[0025] (2) The characteristic peptides are rationally designed, with good sensitivity and stability: The characteristic peptides (Aβ28-42 and Aβ28-40) generated by Lys-N protease hydrolysis are selected. They have strong hydrophilicity and high isoelectric point, and have better ionization efficiency and signal response in mass spectrometry. Compared with the detection of easily aggregated intact proteins, the enzymatically hydrolyzed peptides are more stable and less prone to aggregation, thereby significantly improving the sensitivity and repeatability of detection and realizing reliable quantification of low-abundance Aβ proteins.

[0026] (3) The pretreatment process is simple and efficient: The innovative method involves direct enzymatic digestion on the magnetic beads after immunocapture and direct termination of the reaction with formic acid, eliminating the cumbersome steps such as elution, lyophilization, reconstitution and solid-phase extraction required in traditional methods. This design significantly shortens the pretreatment time, reduces sample loss and operational variability, and improves analytical throughput and the robustness of the overall process.

[0027] (4) The enzyme digestion strategy is safe and efficient: The present invention uses Lys-N enzyme for digestion, which is different from the Trypsin / Lys-C or highly toxic cyanogen bromide (BrCN) used in the prior art. The selected enzyme digestion conditions are mild and safe. The obtained characteristic peptides not only have good hydrophilicity and high ionization efficiency, but also effectively avoid interference with other Aβ subtype peptides, further ensuring the specificity of detection.

[0028] (5) The instrument platform is widely available and easy to promote in clinical practice: The method of this invention is based on ultra-high performance liquid chromatography, which is widely used in the medical system. Triple quadrupole tandem mass spectrometry (UHPLC) The method was developed using an MS / MS platform, rather than relying on expensive and complex nanoliter liquid chromatography-high resolution mass spectrometry systems. This makes the method clinically applicable and cost-effective, facilitating its large-scale application in routine testing laboratories and providing a reliable and accessible detection method for the early screening and diagnosis of Alzheimer's disease. Attached Figure Description

[0029] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is the standard curve for Aβ42; Figure 2 This is the standard curve for Aβ40; Figure 3 The chromatogram of point C1, the calibration minimum point of the Aβ40 calibration curve; Figure 4 The chromatogram of point C1, the calibration minimum point of the Aβ42 calibration curve; Figure 5 This is a schematic diagram of the enzyme-digested peptides of the present invention; Figure 6 This is a schematic diagram of the structure of the characteristic peptide Aβ28-40 in this invention; Figure 7 This is a schematic diagram of the structure of the characteristic peptide Aβ28-42 in this invention; Figure 8 This is a comparison of the signal values ​​of the complete peptides Aβ40 and Aβ42 with the same mass concentration and their corresponding enzymatically digested characteristic peptides Aβ28-40 and Aβ28-42 in Example 3. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0031] Example 1 Step 1: Antibody magnetic bead conjugation 1. Resuspend the magnetic beads in the organic solvent dimethylformamide (DMF) to facilitate removal from the sample vial.

[0032] 2. Before use, vortex for 1-2 minutes to fully resuspend the magnetic beads. Transfer the solution volume corresponding to 20 mg of magnetic beads to a capped test tube.

[0033] 3. Place the test tube on the magnetic rack and let it stand for 4 minutes. Carefully remove the supernatant, being careful not to disturb the magnetic beads.

[0034] 4. Resuspend the magnetic beads in 400 µL of 0.1 M phosphate buffer (pH 7.4).

[0035] 5. Add 400 µL of 0.5 mg / mL anti-Aβ monoclonal antibody 6E10 (purchased from Biolegend), mix thoroughly or vortex, and then add 400 µL of 3 mol / L ammonium sulfate solution.

[0036] 6. Incubate at 37 °C using a rotary mixer for 16-24 hours. Ensure the magnetic beads do not settle during incubation.

[0037] 7. Place the test tube on the magnetic rack for 2 minutes. Gently invert the magnetic rack twice to ensure that any magnetic beads adhering to the tube cap are collected. Remove the supernatant.

[0038] 8. Wash the coated magnetic beads four times with 1 mL PBS buffer, and store them in PBS solution with 2% BSA for later use. Resuspend the magnetic beads after each wash and place them on a magnetic rack for 2 minutes.

[0039] Step 2: Immunoenrichment of Samples 1. Take a clean 1.5 mL centrifuge tube and add the reagents in the following order and volume to prepare the mixture: (1) Clarified plasma obtained by centrifugation at 800 μL; (2) 13 μL 100× protease inhibitor mixture (Halt™ protease inhibitor mixture, ThermoFisher, 78430). (3) 26 μL 10% CHAPS (can be replaced with other detergents such as: Tween20, Igepal CA630, SDS, CHAPSO, Triton X-100); (4) 50 μL 10× PBS; (5) 20 μL containing internal standard (8 ng / mL) 15 N-Aβ40, 4 ng / mL 15 (N-Aβ42), a working solution using water and acetonitrile as solvents.

[0040] 2. Use a rotary mixer to thoroughly mix the above mixture.

[0041] 3. Add 40 μL of the magnetic bead suspension coated with Aβ monoclonal antibody prepared in step one to the mixture.

[0042] 4. Place the centrifuge tubes on a rotary mixer and incubate in a refrigerator (~4°C) for 6 hours to ensure that Aβ is fully captured.

[0043] Step 3: Washing the sample 1. Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed to the tube wall and the solution becomes clear.

[0044] 2. Carefully aspirate the supernatant, being careful not to disturb the magnetic beads.

[0045] 3. Remove the centrifuge tube from the magnetic rack, add 1 mL of 1×PBS, and vortex mix to resuspend the magnetic beads.

[0046] 4. Repeat steps 1 and 2 to complete a total of 3 PBS washes.

[0047] 5. Using 1 mL of 50 mM ammonium bicarbonate (ABC) solution, resuspend, magnetically separate, and discard the supernatant as described above to complete one washing cycle.

[0048] Step 4: Enzymatic digestion and mass spectrometry analysis on magnetic beads 1. Add 50 μL of 50 mM ABC and 1 μL of 100 ng / μL Lys-N metalloproteinase solution to the dried sample. 2. Place the mixture in a constant temperature shaker and incubate at 37°C and 1400 RPM for 4 hours.

[0049] Characteristic peptides obtained by enzymatic digestion of Aβ40 and Aβ42 with Lys-N metalloproteinase are as follows: Figure 5 As shown in Table 4, the ion pair information of the characteristic peptides is presented.

[0050] The amino acid sequence of the complete Aβ40 peptide is as follows: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO.1); The amino acid sequence of the characteristic peptide (Aβ28-40) obtained after Aβ40 is digested with Lys-N enzyme is as follows: KGAIIGLMVGGVV (SEQ ID NO.2) has a quantitative ion concentration of 548.8++.

[0051] The amino acid sequence of the complete Aβ42 peptide is as follows: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO.3); The amino acid sequence of the characteristic peptide (Aβ28-42) obtained after digestion of Aβ42 with Lys-N enzyme is as follows: KGAIIGLMVGGVVIA (SEQ ID NO.4) has a quantitative ion concentration of 598.4++.

[0052] 3. Add 5 μL of formic acid (FA) to the digested sample and vortex to terminate the enzymatic reaction.

[0053] 4. Centrifuge the enzyme digest at 14000 RPM for 10 min, place the centrifuge tube on a magnetic rack, let it stand for 2 minutes, transfer the supernatant to a sample vial, and take 20 μL for LC-MS / MS analysis.

[0054] Liquid chromatography conditions: Chromatographic column: Reversed-phase chromatographic column; Column temperature: 50℃; Mobile phase A: 0.1% aqueous solution of formic acid; Mobile phase B: 0.1% formic acid in acetonitrile solution; Injection volume: 10 μL; Reversed-phase column: diphenyl, 80 Å, 2.6 µm, 2.1 mm × 50 mm; The gradient elution conditions are shown in Table 1 below: Table 1 Elution gradient table

[0055] Note: Gradient elution in different liquid phase systems needs to be optimized.

[0056] The mass spectrometry parameters are shown in Table 2 below (taking RZ-500 as an example): Table 2 Mass Spectrometry Parameters

[0057] Note: Ion source parameters for different models of mass spectrometers need to be optimized.

[0058] The ion pair parameters are shown in Table 3 below: Table 3 Characteristic peptides and their... 15 N internal standard ion pair information

[0059] Example 2 1. Aβ40 is prepared with 6 concentrations of standard: 20, 50, 100, 200, 400, and 1200 pg / ml; Aβ42 is prepared with 6 concentrations of standard: 10, 25, 50, 100, 200, and 600 pg / ml.

[0060] The above standards were analyzed using the IP-LC-MS / MS method, and the isotope internal standard quantification method was employed. A calibration curve was established with the concentration ratio of the standards as the x-axis and the peak area ratio of the standards to the internal standard as the y-axis. The results are as follows. Figure 1 and Figure 2 As shown. Aβ40 ( Figure 2 ) and Aβ42 ( Figure 1 The linear fitting equations within their respective concentration ranges showed good linearity, with correlation coefficients above 0.99.

[0061] 2. Calculate the deviation between the actual and theoretical values ​​of the detected standard samples, as shown in Table 4 below: Table 4. Relative Deviation between Actual and Theoretical Values

[0062] The deviations of the measured results were all within 15%. This indicates that the method based on immunoprecipitation to extract Aβ40 and Aβ42 from plasma and then digesting them with Lys-N enzyme is feasible and reliable.

[0063] Furthermore, at the lowest point Cal 1 of the detection calibrator, the signal-to-noise ratio of both targets was greater than 10 ( ). Figure 3 / 4), which can meet the testing requirements.

[0064] Example 3 1. Different enzymes were used to cleave the intact peptides of Aβ40 and Aβ42, and different characteristic peptides were obtained by hydrolysis. The comparison of the physicochemical properties of each characteristic peptide is shown in Table 5 below: Table 5. Physicochemical properties of different characteristic peptide segments

[0065] As shown in Table 5 above, compared with the characteristic peptides obtained by other types of enzymes or chemical cleavage in the prior art, the characteristic peptides obtained by enzymatic cleavage of the intact Aβ40 and Aβ42 peptides using Lys-N protease in this invention have lower hydrophobicity and higher isoelectric point. The characteristic peptides (Aβ28-42 and Aβ28-40) generated by Lys-N protease hydrolysis in this invention have strong hydrophilicity and high isoelectric point, resulting in better ionization efficiency and signal response in mass spectrometry.

[0066] 2. For example Figure 6 As shown, this is the characteristic peptide Aβ in this invention. 28-40 Structural diagram; such as Figure 7 As shown, this is the characteristic peptide Aβ in this invention. 28-42 A structural diagram.

[0067] Depend on Figure 6 and Figure 7 It can be seen that the characteristic peptide Aβ obtained by Lys-N cleavage in this invention 28-40 and Aβ 28-42 It contains lysine and has an amino group on its side chain, carrying two charges, which further proves that the characteristic peptide of the present invention does indeed have higher ionization efficiency.

[0068] 3. Using the liquid chromatography-tandem mass spectrometry method described in Example 1, intact peptides Aβ40 and Aβ42 of the same mass concentration and their corresponding enzymatically digested characteristic peptides Aβ were analyzed. 28-40 and Aβ 28-42 Perform detection, record signal values, and display them. Figure 8 In the middle. By Figure 8 It can be seen that the characteristic peptides of the present invention have significantly higher detection signal values.

[0069] Compared to detecting easily aggregated intact proteins, the characteristic peptides digested by Lys-N enzymes in this invention are more stable and less prone to aggregation, thereby significantly improving the sensitivity and repeatability of detection and achieving reliable quantification of low-abundance Aβ proteins.

[0070] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A mass spectrometry method for determining the ratio of Aβ protein 42 / 40 in human plasma, characterized in that, Includes the following steps: S1, take anti β Amyloid antibody, coupled with magnetic beads, get Aβ protein antibody coated magnetic beads; S2. Add a mixed internal standard solution of Aβ40 and Aβ42 to a human plasma sample, then add magnetic beads coated with the Aβ protein antibody and mix and incubate to perform immunologic capture of Aβ protein and internal standard Aβ protein in the human plasma sample to obtain Aβ protein-magnetic bead complex. S3. The Aβ protein on the washed Aβ protein-magnetic bead complex is directly enzymatically digested using Lys-N metalloproteinase, so that the captured natural Aβ protein and the internal standard Aβ protein are respectively enzymatically digested to produce corresponding characteristic peptides, wherein the characteristic peptide used to quantify Aβ42 is KGAIIGLMVGGVVIA, and the characteristic peptide used to quantify Aβ40 is KGAIIGLMVGGVV. S4. Detect the characteristic peptide signals generated by the hydrolysis of natural Aβ protein and the isotopically labeled characteristic peptide signals generated by the internal standard Aβ protein by liquid chromatography-tandem mass spectrometry; calculate the concentrations of Aβ42 and Aβ40 in human plasma samples based on the ratio of the natural characteristic peptide signals to their corresponding isotopically labeled characteristic peptide signals, and thus obtain the ratio of Aβ42 to Aβ40.

2. The mass spectrometry method of claim 1, wherein, Step S1 specifically includes: first resuspending the magnetic beads with phosphate buffer, adding anti-Aβ antibody, mixing thoroughly, then adding ammonium sulfate solution, and incubating; the incubation temperature is 30~40 ℃, and the time is 16-24 h.

3. The mass spectrometry method of claim 1, wherein, In step S2, the internal standard of Aβ40 is 15 N-Aβ40, the internal standard of Aβ42 is 15 N-Aβ42; in the mixed internal standard solution, 15 the final concentration of N-Aβ40 is 50-400 pg / mL, 15 the final concentration of N-Aβ42 is 25-400 pg / mL.

4. The mass spectrometry method of claim 1, wherein, In step S2, the mixed incubation system further includes a mixture of protease inhibitors; and a detergent selected from at least one of CHAPS, Tween20, Igepal CA630, CHAPSO, SDS, and Triton X-100.

5. The mass spectrometry method of claim 1, wherein, In step S2, the temperature for the mixed incubation is 3~10 ℃ and the time is 1~8 h.

6. The mass spectrometry method of claim 1, wherein, In step S3, the washing includes resuspending the Aβ protein-magnetic bead complex with PBS 2 to 5 times, followed by resuspending with ammonium bicarbonate solution.

7. The mass spectrometry method of claim 1, wherein, Step S3 specifically includes: adding ammonium bicarbonate solution to the Aβ protein-magnetic bead complex, then adding Lys-N metalloproteinase solution, mixing and incubating with shaking to digest; after digestion, adding formic acid to terminate the enzymatic reaction, centrifuging the reaction product and taking the supernatant for subsequent LC-MS / MS analysis.

8. The mass spectrometry method according to claim 7, characterized in that, The concentration of the ammonium bicarbonate solution is 25~100mM; And / or, the concentration of the Lys-N metalloproteinase solution is 1~10 ng / μL; And / or, the temperature of the oscillation incubation is 25~45℃, the rotation speed is 1000~1400 rpm, and the time is 1~8 h; And / or, the centrifugation speed is 10000~14000 rpm, and the time is 5~15 min.

9. The mass spectrometry method according to claim 1, characterized in that, In step S3, the quantitative ion of the characteristic peptide of Aβ40 is 548.8++; the quantitative ion of the characteristic peptide of Aβ42 is 598.4++.

10. The mass spectrometry method according to claim 1, characterized in that, In step S4, when performing liquid chromatography-tandem mass spectrometry analysis, the chromatographic column is a reversed-phase chromatographic column, and the column temperature is 40~60℃; Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is an acetonitrile solution of 0.1% formic acid.

Images