Synchronous detection method for A53T mutant type and wild type alpha-synuclein

Abstract

The invention relates to a synchronous detection method of A53T mutant type and wild type alpha-synuclein, and belongs to the technical field of protein analysis and detection. According to the detection method disclosed by the invention, the A53T mutant type and wild type alpha-synuclein is subjected to liquid-phase mass spectrometry detection through the A53T mutant type and wild type characteristic peptide fragments. The mass spectrum parent ions and the mass spectrum daughter ions of the characteristic peptide fragment have better specificity and sensitivity, the accuracy of the detection method is improved, the mass spectrum parent ions and the mass spectrum daughter ions of the characteristic peptide fragment have stronger response signals, meanwhile, the signal-to-noise ratio (S / N) of the target peptide fragment is increased by more than 5 times through concentration, and the detection accuracy is improved. The accuracy, repeatability and sensitivity of the method for detecting the protein in the biological matrix can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis and detection, and particularly to a method for simultaneously detecting A53T mutant and wild-type α-synuclein. Background Art

[0002] α-synuclein is a highly conserved presynaptic soluble protein, and its abnormal aggregation is closely related to the pathological processes of synucleinopathies such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Research shows that the imbalance of the homeostasis of wild-type α-synuclein (WTα-synuclein) is a common mechanism of neurodegeneration, and the A53T mutant α-synuclein (i.e., alanine at position 53 is replaced by threonine) has become a key driver of familial Parkinson's disease due to its significantly enhanced oligomerization ability. In the progression of related diseases, the dynamic change of the concentration ratio of A53T mutant and wild-type α-synuclein directly affects the pathological phenotype. Therefore, developing a technical means that can simultaneously detect and accurately quantify the two is of great value for analyzing disease mechanisms, evaluating therapeutic targets, and developing companion diagnostic tools.

[0003] Currently, the detection methods widely used in preclinical studies are mainly based on immunological principles (such as ELISA, Simoa ultrasensitive detection platform), which rely on antibodies against specific epitopes to achieve the recognition of target proteins. However, such methods have the following defects when simultaneously distinguishing A53T mutants from wild-type α-synuclein:

[0004] Epitope cross-reactivity: The A53T mutation only results in a single amino acid substitution, and it is difficult for traditional antibodies to distinguish between mutant and wild-type with highly similar spatial conformations. Even when using so-called "mutation-specific antibodies", false positive / false negative signals may still be generated due to the interference of the epitope microenvironment;

[0005] Limited dynamic range: When A53T mutant and wild-type α-synuclein coexist, the competitive binding of the two to antibodies will lead to quantitative deviation. Especially in the scenario where a low-abundance mutant (such as accounting for <1% of the total) coexists with a high-abundance wild-type, the detection accuracy significantly decreases;

[0006] High cost of multiplex detection: If two subtypes need to be simultaneously detected, usually two independent antibody systems need to be developed and reaction conditions need to be optimized, which significantly increases the reagent development and experimental verification costs.

[0007] Therefore, there is an urgent need to develop a highly specific and highly compatible mass spectrometry detection method that can synchronously distinguish and accurately quantify A53T mutant and wild-type α-synuclein in a single analysis, while overcoming the problems of ion interference and calibration in the coexisting system to meet the urgent needs for dynamic monitoring of protein subtypes in basic research and clinical translation. Summary of the Invention

[0008] The object of the present invention is to overcome the deficiencies of the prior art and provide a synchronous detection method for A53T mutant and wild-type α-synuclein. The detection method of the present invention can detect A53T mutant and wild-type α-synuclein in a sample, and achieve synchronous distinction and accurate quantification of the two.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect, the present invention provides a synchronous detection method for A53T mutant and wild-type α-synuclein, and the synchronous detection method includes the following steps:

[0011] S1 Pretreat the sample:

[0012] S2 The pretreated sample is detected by a liquid-phase mass spectrometer to obtain the response signals of the characteristic peptides of A53T mutant and wild-type α-synuclein in the liquid-phase mass spectrometer;

[0013] Among them, the characteristic peptide 1 of A53T mutant α-synuclein is EGVVHGVTTVAEK;

[0014] The characteristic peptide 2 of wild-type α-synuclein is EGVVHGVATVAEK;

[0015] S3 Convert the response signals obtained by the characteristic peptides in the liquid-phase mass spectrometer into the contents of A53T mutant and wild-type α-synuclein in the sample according to the standard curve.

[0016] The present invention obtained different characteristic peptide fragments of wild-type and A53T mutant α-synuclein by trypsin digestion, that is, the characteristic peptide fragment 1 of A53T mutant α-synuclein is EGVVHGVTTVAEK; the characteristic peptide fragment 2 of wild-type α-synuclein is EGVVHGVATVAEK; the A53T mutant and wild-type α-synuclein can be used for liquid chromatography-mass spectrometry detection through the characteristic peptide fragments, so as to carry out separation and quantification. The mass spectrometry precursor ions and product ions of the characteristic peptide fragments of the present invention have better specificity and sensitivity, avoiding the interference of many subtypes with similar structures to wild-type and A53T mutant α-synuclein in the biological matrix to the detection, which is beneficial to improving the accuracy of the detection method, and the mass spectrometry precursor ions and product ions of the characteristic peptide fragments have stronger response signals. At the same time, the present invention improves the signal-to-noise ratio (S / N) of the target peptide fragment by more than 5 times through concentration, and can improve the accuracy, repeatability and sensitivity of the protein detection method in the biological matrix.

[0017] As a preferred embodiment of the first aspect, the sample pretreatment method in step S1 includes the following steps:

[0018] I Ultrafilter the sample and collect the filtrate;

[0019] II Add a buffer solution to the filtrate and incubate with shaking;

[0020] III Add a protein denaturant in step II and incubate;

[0021] IV Add a protein stabilizer in step III, incubate and then lyophilize the sample;

[0022] V Reconstitute the lyophilized sample in step IV with a buffer solution, add trypsin for enzymatic digestion and then lyophilize the sample.

[0023] In the process of studying sample pretreatment, the present invention found that different pretreatment methods will affect the subsequent detection results. Therefore, by optimizing the selection of ultrafiltration and the combination of protein denaturant, protein stabilizer and lyophilization, the detection effect can be improved. In the pretreatment method of the present invention, the signal-to-noise ratio (S / N) of the target peptide fragment is increased by more than 5 times through concentration, and the detection limit (LOD) of the A53T mutant is as low as 0.2 ng / mL, and clinical samples with the proportion of A53T mutant α-synuclein as low as 1% can be accurately detected. Compared with other pretreatment methods, for example, immunoprecipitation method, there is no need for specific antibody magnetic beads and solid-phase extraction columns to concentrate the sample, and the ultrafiltration tube can be used repeatedly, greatly saving costs, saving operation time and improving efficiency.

[0024] As a preferred embodiment of the first aspect of the present invention, the buffer solution is ammonium bicarbonate solution, the protein denaturant is dithiothreitol solution, and the protein stabilizer is iodoacetamide solution.

[0025] The sample described in the present invention is a biological matrix sample, including blood, urine, saliva, milk, tears, cerebrospinal fluid, bile, gastric juice, feces, tissue homogenate, etc.

[0026] The present invention also uses trypsin to cleave A53T mutant α-synuclein and wild-type α-synuclein to generate highly stable characteristic peptide segments, improving detection sensitivity and reproducibility. That is, the characteristic peptide segment 1 of A53T mutant α-synuclein is EGVVHGVTTVAEK; the characteristic peptide segment 2 of wild-type α-synuclein is EGVVHGVATVAEK.

[0027] As a preferred embodiment of the first aspect, the mass spectrometry conditions of the liquid mass spectrometer in step S2 satisfy the following conditions:

[0028] (1) The parent ion of the characteristic peptide segment 1 is 442.6, and the daughter ion is 347.2;

[0029] (2) The declustering voltage of the characteristic peptide segment 1 is 80V, and the collision energy is 24 - 34 eV;

[0030] (3) The parent ion of the characteristic peptide segment 2 is 648.4, and the daughter ion is 347.4;

[0031] (4) The declustering voltage of the characteristic peptide segment 2 is 80V, and the collision energy is 40 - 50 eV.

[0032] As a preferred embodiment of the first aspect, the mass spectrometry conditions of the liquid mass spectrometer in step S2 satisfy the following conditions:

[0033] (1) The parent ion of the characteristic peptide segment 1 is 442.6, and the daughter ion is 347.2;

[0034] (2) The declustering voltage of the characteristic peptide segment 1 is 80V, and the collision energy is 29 eV;

[0035] (3) The parent ion of the characteristic peptide segment 2 is 648.4, and the daughter ion is 347.4;

[0036] (4) The declustering voltage of the characteristic peptide segment 2 is 80V, and the collision energy is 45 eV.

[0037] In the process of studying the sample detection method, the present invention found that different mass spectrometry conditions will affect the subsequent detection results. Therefore, through optimization, it is found that when the declustering voltage of the characteristic peptide segment 1 is 80V and the collision energy is 29 eV, and when the declustering voltage of the characteristic peptide segment 2 is 80V and the collision energy is 45 eV, the peak area is the largest and the effect is the best.

[0038] As a preferred embodiment of the first aspect, the liquid phase conditions of the liquid chromatography-mass spectrometry in step S2 satisfy the following conditions:

[0039] (1) The mobile phase is: aqueous phase 0.1% formic acid aqueous solution, organic phase 0.1% formic acid acetonitrile solution;

[0040] (2) The mobile phase gradient is:

[0041] 0 - 0.50 min, the volume ratio of the aqueous phase to the organic phase is: 95%: 5%;

[0042] 0.50 - 3.00 min, the volume ratio of the aqueous phase to the organic phase is: (60% - 95%): (5% - 40%);

[0043] 3.00 - 3.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 60%): (40% - 85%);

[0044] 3.01 - 4.00 min, the volume ratio of the aqueous phase to the organic phase is: 15%: 85%;

[0045] 4.00 - 4.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 95%): (5% - 85%);

[0046] 4.01 - 5.00 min, the volume ratio of the aqueous phase to the organic phase is: 95%: 5%.

[0047] In the process of researching the sample detection method of the present invention, it is found that different liquid phase conditions will affect the subsequent detection results. Therefore, through optimization, it is found that when the mobile phase is aqueous phase 0.1% formic acid aqueous solution and organic phase 0.1% formic acid acetonitrile solution, the peak area is the largest and the effect is the best.

[0048] As a preferred embodiment of the first aspect, it further includes the step of detecting the total amount of α-synuclein.

[0049] As a preferred embodiment of the first aspect, the step of detecting the total amount of α-synuclein is:

[0050] a The pretreated sample is detected by a liquid chromatography-mass spectrometer to obtain the response signal of the characteristic peptide segment of α-synuclein in the liquid chromatography-mass spectrometer;

[0051] Among them, the characteristic peptide segment 3 of α-synuclein is QGVAEAAGK; and / or, the characteristic peptide segment 4 of α-synuclein is TVEGAGSIAAATGFVK;

[0052] b The corresponding signals of the characteristic peptide segments obtained in step a in the liquid chromatography-mass spectrometer are respectively converted into the content of α-synuclein in the sample according to the standard curve.

[0053] As a preferred embodiment of the first aspect, the mass spectrometry conditions for detecting the total amount of α-synuclein satisfy the following conditions:

[0054] (1) The parent ion of the characteristic peptide segment 3 is 415.7, and the daughter ion is 346.2;

[0055] (2) The declustering voltage of the characteristic peptide segment 3 is 80 V, and the collision energy is 24 - 34 eV;

[0056] (3) The parent ion of the characteristic peptide segment 4 is 739.9, and the daughter ion is 173.1;

[0057] (4) The declustering voltage of the characteristic peptide segment 4 is 80 V, and the collision energy is 40 - 50 eV.

[0058] As a preferred embodiment of the first aspect, the mass spectrometry conditions for detecting the total amount of α-synuclein satisfy the following conditions:

[0059] (1) The parent ion of the characteristic peptide segment 3 is 415.7, and the daughter ion is 346.2;

[0060] (2) The declustering voltage of the characteristic peptide segment 3 is 80 V, and the collision energy is 29 eV;

[0061] (3) The parent ion of the characteristic peptide segment 4 is 739.9, and the daughter ion is 173.1;

[0062] (4) The declustering voltage of the characteristic peptide segment 4 is 80 V, and the collision energy is 45 eV.

[0063] In the process of researching the sample detection method of the present invention, it is found that different mass spectrometry conditions will affect the subsequent detection results. Therefore, through optimization, it is found that when the declustering voltage of the characteristic peptide segment 3 is 80 V and the collision energy is 29 eV, and when the declustering voltage of the characteristic peptide segment 4 is 80 V and the collision energy is 45 eV, the peak area is the largest and the effect is the best.

[0064] In the second aspect, the present invention provides a characteristic peptide segment for detecting A53T mutant and wild-type α-synuclein. The amino acid sequence of the characteristic peptide segment for detecting A53T mutant α-synuclein is as shown in SEQ ID NO: 1, and / or, the amino acid sequence of the characteristic peptide segment for detecting wild-type α-synuclein is as shown in SEQ ID NO: 2.

[0065] In the third aspect, the present invention provides a characteristic peptide segment for detecting α-synuclein, and the amino acid sequence of the characteristic peptide segment is as shown in any one of SEQ ID NOs: 3 - 4.

[0066] In a fourth aspect, the present invention provides the use of the characteristic peptide segments described in the second aspect in a method for synchronously detecting A53T mutant and wild-type α-synuclein, or the use of the characteristic peptide segments described in the third aspect in a method for detecting the total content of α-synuclein.

[0067] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0068] 1. Breakthrough in solving the technical barrier of synchronous detection of A53T mutant / wild-type α-synuclein

[0069] By using an enzymatic digestion strategy (trypsin) to generate peptide segments containing the A53T site, it is not necessary to rely on high-resolution mass spectrometry equipment (such as Orbitrap), and baseline separation can be achieved on a conventional triple quadrupole mass spectrometer (QqQ), reducing the detection cost by more than 60%.

[0070] 2. Achieving high-precision quantification in a coexisting system

[0071] Based on the design of non-overlapping characteristic ion pairs, spectral signal cross-interference is effectively avoided, and the quantitative repeatability (RSD) of A53T mutant and wild-type α-synuclein reaches 15% (n = 3) respectively.

[0072] 3. Ultra-sensitive detection of low-abundance mutant proteins

[0073] Combined with ultrafiltration technology, the signal-to-noise ratio (S / N) of the target peptide segment is increased by more than 5 times, and the limit of detection (LOD) of A53T mutant α-synuclein is as low as 0.2 ng / mL, enabling accurate detection of clinical samples with the proportion of A53T mutant α-synuclein as low as 1%.

[0074] 4. Strong compatibility and easy clinical translation

[0075] Based on the standardized process of a conventional LC-QqQ-MS platform (chromatographic gradient 5 min / sample), it supports a sample throughput of 12 samples per hour. With a pre-installed kit and an automated analysis module, it can be directly deployed in a clinical laboratory, saving 90% of the labor cost compared with traditional customized mass spectrometry solutions (requiring professional operators).

[0076] 5. Expandability for multi-scenario applications

[0077] It is applicable to various biological samples such as cerebrospinal fluid, plasma, serum, and exosomes. The quantitative linear range of A53T mutant α-synuclein is 0.2 ng / mL - 80 ng / mL (R 2 > 0.99), covering the needs of clinical diagnosis (low concentration) and pathological research (high concentration), providing a general technology platform for the precision medicine of neurodegenerative diseases.

[0078] 6. Low-cost pretreatment

[0079] Without specific antibody magnetic beads and solid-phase extraction columns for sample concentration, the ultrafiltration tubes can be reused, greatly saving costs. The pretreatment cost for each sample is no more than 10 yuan. Description of the Drawings

[0080] Figure 1 It is the standard curve graph for the characteristic peptide segment 1 EGVVHGVTTVAEK (442.6→347.2);

[0081] Figure 2 It is the standard curve graph for the characteristic peptide segment 2 EGVVHGVATVAEK (648.4→347.4);

[0082] Figure 3 It is the standard curve graph for the characteristic peptide segment 3 QGVAEAAGK (415.7→346.2);

[0083] Figure 4 It is the standard curve graph for the characteristic peptide segment 4 TVEGAGSIAAATGFVK (739.9→173.1);

[0084] Figure 5 It is the schematic diagram of the detection results of the separation of the characteristic peptide segment 1 EGVVHGVTTVAEK (442.6→347.2), the characteristic peptide segment 2 EGVVHGVATVAEK (648.4→347.4), the characteristic peptide segment 3 QGVAEAAGK (415.7→346.2), and the characteristic peptide segment 4 TVEGAGSIAAATGFVK (739.9→173.1) from the sample. Detailed Embodiments

[0085] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0086] Example 1

[0087] This example provides a synchronous detection method for A53T mutant and wild-type α-synuclein, including the following steps:

[0088] S1 Pretreat the sample, including the following steps:

[0089] I Ultrafilter the sample and collect the filtrate;

[0090] II Add a buffer solution to the filtrate and incubate with shaking;

[0091] III Add a protein denaturant solution in step II and incubate;

[0092] IV Add a protein stabilizer in step III, incubate, and then lyophilize the sample;

[0093] Reconstitute the lyophilized sample in step IV with buffer solution for V, add trypsin for enzymatic digestion, and then lyophilize the sample.

[0094] S2 Detect the pretreated sample by liquid chromatography-mass spectrometry (LC-MS) to obtain the response signals of characteristic peptides 1 and 2 of A53T mutant and wild-type α-synuclein, and characteristic peptides 3 and 4 for detecting the total content of α-synuclein in the LC-MS;

[0095] Among them, the characteristic peptide 1 of A53T mutant α-synuclein is EGVVHGVTTVAEK (SEQ ID NO: 1);

[0096] The characteristic peptide 2 of wild-type α-synuclein is EGVVHGVATVAEK (SEQ ID NO: 2);

[0097] The characteristic peptide 3 of α-synuclein is QGVAEAAGK (SEQ ID NO: 3); and / or, the characteristic peptide 4 of α-synuclein is TVEGAGSIAAATGFVK (SEQ ID NO: 4);

[0098] The mass spectrometry conditions of the liquid chromatography-mass spectrometry in step S2 meet the following conditions:

[0099] (1) The parent ion of the characteristic peptide 1 is 442.6, and the daughter ion is 347.2;

[0100] (2) The declustering voltage of the characteristic peptide 1 is 80 V, and the collision energy is 24 - 34 eV;

[0101] (3) The parent ion of the characteristic peptide 2 is 648.4, and the daughter ion is 347.4;

[0102] (4) The declustering voltage of the characteristic peptide 2 is 80 V, and the collision energy is 40 - 50 eV;

[0103] (5) The parent ion of the characteristic peptide 3 is 415.7, and the daughter ion is 346.2;

[0104] (6) The declustering voltage of the characteristic peptide 3 is 80 V, and the collision energy is 24 - 34 eV;

[0105] (7) The parent ion of the characteristic peptide 4 is 739.9, and the daughter ion is 173.1;

[0106] (8) The declustering voltage of the characteristic peptide 4 is 80 V, and the collision energy is 40 - 50 eV;

[0107] The mass spectrometry conditions of the liquid chromatography-mass spectrometry in step S2 also meet the following conditions:

[0108] (1) Mass spectrometer: SCIEX Triple Quad 6500+;

[0109] (2) Ion source: Electrospray ionization source (ESI);

[0110] (3) Ion source temperature (°C): 550;

[0111] (4) Entrance voltage (KV): 10;

[0112] (5) Gas1 (L·h -1 ): 50;

[0113] (6) Gas2 (L·h -1 ): 50;

[0114] (7) Curtain Gas (L·h -1 ): 35;

[0115] (8) Collision Gas (L·h -1 ): Medium;

[0116] (9) Ionization mode: Positive ion;

[0117] (10) Signal acquisition mode: MRM.

[0118] The liquid phase conditions of the liquid chromatography - mass spectrometer in step S2 satisfy the following conditions:

[0119] (1) Mobile phase: Aqueous phase is 0.1% formic acid aqueous solution, organic phase is 0.1% formic acid acetonitrile solution;

[0120] (2) Mobile phase gradient is:

[0121] 0 - 0.50 min, the volume ratio of the aqueous phase to the organic phase is: 95%:5%;

[0122] 0.50 - 3.00 min, the volume ratio of the aqueous phase to the organic phase is: (60% - 95%):(5% - 40%);

[0123] 3.00 - 3.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 60%):(40% - 85%);

[0124] 3.01 - 4.00 min, the volume ratio of the aqueous phase to the organic phase is: 15%:85%;

[0125] 4.00 - 4.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 95%):(5% - 85%);

[0126] 4.01 - 5.00 min, the volume ratio of the aqueous phase to the organic phase is: 95%:5%;

[0127] (3) Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7 μm (2.1×100 mm);

[0128] (4) Column temperature: 40 °C; Autosampler temperature: 15 °C;

[0129] (5) Flow rate: 0.3 mL / min;

[0130] (6) Injection volume: 10 μL.

[0131] S3 Convert the response signals obtained for the characteristic peptide segments in the liquid mass spectrometer into A53T mutant and wild-type α-synuclein respectively according to the standard curve, as well as the content of α-synuclein in the sample.

[0132] Preferably, the buffer solution is an ammonium bicarbonate solution, the protein denaturant is a dithiothreitol solution, and the protein stabilizer is an iodoacetamide solution.

[0133] Preferably, the declustering voltage of the characteristic peptide segment 1 is 80 V, and the collision energy is 29 eV;

[0134] Preferably, the declustering voltage of the characteristic peptide segment 2 is 80 V, and the collision energy is 45 eV;

[0135] Preferably, the declustering voltage of the characteristic peptide segment 3 is 80 V, and the collision energy is 29 eV;

[0136] Preferably, the declustering voltage of the characteristic peptide segment 4 is 80 V, and the collision energy is 45 eV.

[0137] I. Experimental method:

[0138] 1. Preparation of main reagents

[0139] 1.1 Wild-type α-synuclein stock solution: Take 100 μg of wild-type α-synuclein standard, dissolve it in 1 mL of pure water to 0.1 mg / mL as the stock solution, and store it at -20 °C after aliquoting.

[0140] 1.2 A53T mutant α-synuclein stock solution: Take 100 μg of A53T mutant α-synuclein standard, dissolve it in 1 mL of pure water to 0.1 mg / mL as the stock solution, and store it at -20 °C after aliquoting.

[0141] 1.3 Wild-type and A53T mutant α-synuclein intermediate working solution: Take 10 μL each of the wild-type α-synuclein and A53T mutant α-synuclein stock solutions, add 980 μL of pure water and dissolve to 1 μg / mL as the intermediate working solution. After aliquoting, store at -20 °C.

[0142] 1.4 A53T mutant α-synuclein intermediate working solution: Take 10 μL of the A53T mutant α-synuclein stock solution, add 990 μL of pure water and dissolve to 1 μg / mL as the stock solution. After aliquoting, store at -20 °C.

[0143] 1.5 Wild-type α-synuclein intermediate working solution: Take 10 μL of the wild-type α-synuclein stock solution, add 990 μL of pure water and dissolve to 1 μg / mL as the intermediate working solution. After aliquoting, store at -20 °C.

[0144] 1.4 Aqueous phase preparation: Mix formic acid and water in a volume ratio of 1:1000, and prepare according to this ratio when in use.

[0145] 1.5 Organic phase preparation: Mix formic acid and acetonitrile in a volume ratio of 1:1000, and prepare according to this ratio when in use.

[0146] 1.6 Standard curve preparation: Dilute the intermediate working solutions of wild-type and A53T mutant α-synuclein with a surrogate biological matrix (6% HSA); the specific dilution method is shown in Table 1, and prepare according to the ratio when in use.

[0147] 1.7 Quality control sample preparation: Dilute the intermediate working solutions of wild-type and A53T mutant α-synuclein with a surrogate biological matrix, and the specific dilution method is shown in Table 2, and prepare according to the ratio when in use.

[0148] Table 1 Standard curve sample preparation table

[0149]

[0150] Table 2 Quality control sample preparation table

[0151]

[0152] 2. Determination of sample pretreatment method:

[0153] 2.1 Different pretreatment methods

[0154] Pretreatment method 1:

[0155] (1) Take 500 μL of the sample in a 50 kD cut-off ultrafiltration tube and centrifuge (2000 g / min, 20 min), and collect the filtrate;

[0156] (2) Add 50 uL of 100 mM ammonium bicarbonate solution and incubate with shaking at 90 °C for 25 min;

[0157] (3) Add 20 uL of 10 mM dithiothreitol (DTT) solution and incubate at 60 °C for 1 h;

[0158] (4) Add 40 uL of 100 mM iodoacetamide solution, incubate in the dark at room temperature for 30 min, and lyophilize the sample;

[0159] (5) Reconstitute with 200 uL of 100 mM ammonium bicarbonate solution, add 50 uL of 0.2 ug / uL trypsin, mix well, incubate overnight at 37 °C, and lyophilize the sample.

[0160] In this example, the characteristic peptide segments of wild-type and A53T mutant α-synuclein hydrolyzed by trypsin are as follows:

[0161] The amino acid sequence of the characteristic peptide segment of A53T mutant α-synuclein is: EGVVHGVTTVAEK (denoted as characteristic peptide segment 1, SEQ ID NO: 1).

[0162] The amino acid sequence of the characteristic peptide segment of wild-type α-synuclein is: EGVVHGVATVAEK (denoted as characteristic peptide segment 2, SEQ ID NO: 2).

[0163] QGVAEAAGK (denoted as characteristic peptide segment 3, SEQ ID NO: 3) and TVEGAGSIAAATGFVK (denoted as characteristic peptide segment 4, SEQ ID NO: 4) are used to detect the total content of α-synuclein.

[0164] Pretreatment method 2:

[0165] The difference between pretreatment method 2 and pretreatment method 1 is that in step (2), incubating with shaking at 90 °C for 25 min is replaced by standing at room temperature for 10 min.

[0166] Pretreatment method 3:

[0167] The difference between pretreatment method 3 and pretreatment method 1 is that in step (4), precipitating the protein with acetonitrile is used to replace lyophilizing the sample.

[0168] Pretreatment method 4:

[0169] The difference between pretreatment method 4 and pretreatment method 1 is that in step (5), enzymatic digestion for 3 - 4 h is used to replace enzymatic digestion overnight.

[0170] Pretreatment method 5:

[0171] The difference between the pretreatment method 5 and the pretreatment method 1 is that in step (5), incubation at room temperature is used to replace incubation at 37°C.

[0172] 2.2 Influence of different pretreatment methods on the detection results

[0173] Since different pretreatment methods have a great influence on the detection results of samples, pretreatment methods 1-5 were compared, and the results are shown in the following table:

[0174] Table 3 Comparison of the detection areas of characteristic peptide segments in different pretreatment methods

[0175] Pretreatment method Peak area of characteristic peptide segment 1 Peak area of characteristic peptide segment 2 Pretreatment method 1 2527004 225960 Pretreatment method 2 2207185 206867 Pretreatment method 3 2067369 183554 Pretreatment method 4 1445714 116433 Pretreatment method 5 1687667 150675

[0176] As can be seen from the above table, when the sample is treated with the pretreatment method 1, the peak areas of the characteristic peptide segments 1 and 2 are the largest. Therefore, the pretreatment method 1 was finally determined as the sample pretreatment method of the present invention.

[0177] 2.3 Investigation of the stability of the pretreated samples

[0178] 2.3.1 Long-term storage stability of samples

[0179] Take surrogate biological matrix to prepare 3 concentration quality control samples of LQC, MQC, and HQC. After pretreatment with the above pretreatment method 1, store them frozen at -80°C for 1 month. When injecting samples, dissolve the samples with 100 μL of 0.1% formic acid aqueous solution. Record the peak areas of the target peptide segments, calculate the protein concentration from the standard curve of the same day, and compare it with the labeled concentration. The average deviation of accuracy is within ±15% of the labeled concentration. The investigation results are shown in the following table.

[0180] Table 4 Long-term stability results

[0181]

[0182]

[0183] 2.3.2 Short-term stability of samples placed at room temperature

[0184] Take surrogate biological matrix to prepare 3 concentration quality control samples of LQC, MQC, and HQC. After pretreatment with the above pretreatment method 1, store them at room temperature for 12 hours. When injecting samples, dissolve the samples with 100 μL of 0.1% formic acid aqueous solution. Record the peak areas of the target peptide segments, calculate the protein concentration from the standard curve of the same day, and compare it with the labeled concentration. The average deviation of accuracy is within ±15% of the labeled concentration. The investigation results are shown in the following table.

[0185] Table 5 Short-term stability results

[0186]

[0187]

[0188] As can be seen from Tables 4 and 5 above, the pretreatment method of the present invention also has the effect of improving the stability of the sample. After being stored in a -80°C refrigerator for 1 month and at room temperature for 12 hours, the average deviation of the sample accuracy is within ±15% of the labeled concentration.

[0189] 3. Determination of mass spectrometry chromatographic conditions

[0190] 3.1 Influence of different collision energies on detection results

[0191] The ion information of characteristic peptide segments 1-4 in this embodiment is as follows in the table:

[0192] Table 6 Ion information of peptide segments 1-4

[0193]

[0194] Note: Peptide segments 3 and 4 are used for the quantification of total α-synuclein (including A53T mutant and wild type).

[0195] In LC-MS / MS detection, the collision energy directly affects the loss and fragmentation degree of ions during the process of entering the mass spectrometer, and has an important influence on the method sensitivity. During the optimization of mass spectrometry parameters, the collision energy was optimized, and the candidate conditions are as follows in the table:

[0196] Table 7 Optimization of mass spectrometry parameters for peptide segments 1-4

[0197]

[0198] As can be seen from the above table, when the declustering voltage of characteristic peptide segment 1 is 80V and the collision energy is 29eV, and when the declustering voltage of characteristic peptide segment 2 is 80V and the collision energy is 45eV, the peak area is the largest and the effect is the best.

[0199] 3.2 Other mass spectrometry conditions:

[0200] Mass spectrometer: SCIEX Triple Quad 6500+;

[0201] Ion source: electrospray ionization source (ESI);

[0202] Ion source temperature (°C): 550;

[0203] Entrance voltage (KV): 10;

[0204] Gas1 (L·h -1 ) : 50;

[0205] Gas2 (L·h -1 ) : 50;

[0206] Curtain Gas (L·h -1 ):35;

[0207] Collision Gas (L·h -1 ):Medium;

[0208] Ionization mode: Positive ion;

[0209] Signal acquisition mode: MRM.

[0210] 4. Determination of liquid chromatography conditions

[0211] 4.1 Selection of chromatographic column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm)

[0212] 4.2 Selection of mobile phase: The candidate mobile phase components are as follows in the table:

[0213] Table 8 Candidate mobile phase components

[0214] Candidate conditions Aqueous phase Organic phase Condition 1 0.1% formic acid aqueous solution 0.1% formic acid acetonitrile solution Condition 2 0.1% ammonia water Acetonitrile Condition 3 0.1% formic acid aqueous solution Acetonitrile Condition 4 0.2% formic acid aqueous solution 0.2% formic acid acetonitrile solution

[0215] Table 9 Comparison of the detection areas of characteristic peptide segments under different mobile phase conditions

[0216] Candidate conditions Peak area of characteristic peptide segment 1 Peak area of characteristic peptide segment 2 Condition 1 7683646 5673465 Condition 2 3386522 2754782 Condition 3 6977628 5084357 Condition 4 7458387 5274633

[0217] From the above peak areas, it can be seen that when condition 1 is selected, the peak areas of characteristic peptide segment 1 and characteristic peptide segment 2 are significantly higher than other conditions. Therefore, condition 1 is finally selected as the mobile phase of the present invention.

[0218] 4.3 Other chromatographic conditions

[0219] Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7μm (2.1×100mm);

[0220] Column temperature: 40°C; Autosampler temperature: 15°C;

[0221] Flow rate: 0.3 mL / min;

[0222] Injection volume: 10 μL;

[0223] Aqueous phase: 0.1% formic acid aqueous solution; Organic phase: 0.1% formic acid acetonitrile solution;

[0224] The gradient is shown in the following table.

[0225] Table 10 Mobile phase gradient

[0226]

[0227]

[0228] II. Performance Evaluation of the Detection Method

[0229] 1. Establishment of Standard Curve and Linear Range

[0230] Prepare a series of standard products with concentrations ranging from 0.2 to 80 ng / mL, record the chromatographic peak areas of the target peptide segments in the standard curve samples of the samples, perform linear regression with the peak area y corresponding to its concentration x, obtain the regression equation of the standard curve, and the weighting coefficient is 1 / x 2 , the linear range of the detection method constructed by the present invention is 0.2 - 80 ng / mL, and its standard curve is as Figures 1 - 4 shown, and the linear inspection results are as follows in the table:

[0231] Table 11 Results of Linear Range Inspection

[0232]

[0233] 2. Precision and Accuracy Inspection

[0234] Take the samples of 3 levels of quality control, namely LQC, MQC, and HQC, and inject them after pretreatment; detect for at least two days, with 3 replicates for each concentration in each batch. Requirements: The average accuracy deviation within and between batches is within ±15% of the labeled value; the precision %CV within and between batches is within 15%. The inspection results are shown in the following table.

[0235] Table 12 Results of Precision and Accuracy Inspection

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] III. Application of the Detection Method

[0242] In order to further verify the detection method constructed by the present invention, we prepared a theoretical concentration: wild-type α-synuclein: 8 ng / mL; A53T mutant α-synuclein: 8 ng / mL, and the total concentration was 16 ng / mL for detection.

[0243] The detection steps are as follows:

[0244] 1. Pretreat the above samples prepared with a substitute biological matrix (6% HSA) using Pretreatment Method 1.

[0245] 2. Reconstitute the pretreated samples with 100 μL of 0.1% formic acid aqueous solution.

[0246] 3. Perform LC-MS / MS detection on the samples in Step 2:

[0247] The liquid chromatography conditions are as follows:

[0248] Chromatographic column: Waters ACQUITY UPLC BEH C8 1.7 μm (2.1×100 mm)

[0249] Mobile phase: Aqueous phase: 0.1% formic acid aqueous solution; Organic phase: 0.1% formic acid acetonitrile solution;

[0250] Elution program: As shown in Table 10.

[0251] Column temperature: 40°C; Autosampler temperature: 15°C;

[0252] Flow rate: 0.3 mL / min;

[0253] Injection volume: 10 μL.

[0254] The mass spectrometry conditions are as follows:

[0255] Mass spectrometer: SCIEX Triple Quad 6500+

[0256] Ion source: Electrospray ionization source (ESI)

[0257] Ion source temperature (°C): 550

[0258] Entrance voltage (KV): 10

[0259] Gas1 (L·h -1 ): 50

[0260] Gas2 (L·h -1 ): 50

[0261] Curtain Gas (L·h -1 ): 35

[0262] Collision Gas (L·h -1 ): Medium

[0263] Ionization mode: Positive ion

[0264] Signal acquisition mode: MRM

[0265] The declustering voltage of characteristic peptide fragment 1 is 80 V, and the collision energy is 29 eV; the declustering voltage of characteristic peptide fragment 2 is 80 V, and the collision energy is 45 eV. The declustering voltage of peptide QGVAEAAGK is 80 V, and the collision energy is 29 eV; the declustering voltage of TVEGAGSIAAATGFVK is 80 V, and the collision energy is 45 eV.

[0266] The detection results are as shown in the following table and Figure 5 are as follows:

[0267] Table 13 Detection of α-synuclein in samples

[0268]

[0269] From Table 13 and Figure 5 it can be seen that the detection method constructed by the present invention can separate and quantitatively detect A53T mutant and wild-type α-synuclein in the sample, and at the same time can also detect the total content of α-synuclein through peptide fragments QGVAEAAGK and TVEGAGSIAAATGFVK. Moreover, the detection method of the present invention can complete the detection within a short time (5 min), greatly shortening the detection time and improving the detection efficiency.

[0270] The synchronous detection and quantification method for A53T mutant and wild-type α-synuclein provided by the present invention, with its advantages of high specificity, high sensitivity and good reproducibility for calibration, can be widely applied to the following scenarios:

[0271] 1. Precise diagnosis and subtype differentiation of neurodegenerative diseases

[0272] Application scenario: For the early differential diagnosis of synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), by detecting the concentration ratio of A53T mutant and wild-type α-synuclein in the blood or cerebrospinal fluid of patients, differentiating sporadic diseases from familial mutation-related subtypes;

[0273] Technical adaptability: The detection limit of the method reaches the nanomolar level (<0.02 nM), which can effectively capture low-abundance mutant α-synuclein (such as the A53T mutant accounting for <1% of the total amount), significantly improving the detection rate of rare mutation cases.

[0274] 2. Dynamic monitoring of the efficacy of targeted therapeutic drugs

[0275] Application scenario: In the clinical trial of α-synuclein aggregation inhibitors, synchronously quantify the concentration changes of A53T mutant and wild-type proteins after drug administration, and evaluate the targeting efficiency of the drug on specific subtypes;

[0276] Technical adaptability: High sensitivity, high specificity, accurately quantify A53T mutant and wild-type α-synuclein. As a detection method for PD indicators of α-synuclein aggregation inhibitors, it helps in the transformation of Parkinson's drugs.

[0277] 3. Pathological mechanism research and biomarker development

[0278] Application scenario: Analyze the dynamic interaction between A53T mutant and wild-type α-synuclein in body fluids (such as cerebrospinal fluid, exosomes), and reveal the molecular pathway by which the mutant promotes protein aggregation;

[0279] Technical adaptability: The non-overlapping characteristic ion pair design can avoid spectral interference in coexisting systems, accurately obtain the absolute concentration data of the two subtypes, and support mechanism modeling based on concentration gradients.

[0280] 4. Early-onset risk assessment and genetic counseling for hereditary Parkinson's disease

[0281] Application scenario: Regularly monitor the protein levels of asymptomatic individuals carrying the A53T mutation in the SNCA gene (encoding α-synuclein) to assess their risk of pathological progression;

[0282] Technical adaptability: Standardized operation procedures and automated data analysis modules support large-scale population screening, reducing the operational complexity of clinical laboratories.

[0283] 5. Development and commercialization of companion diagnostic kits

[0284] Application scenario: Integrate the ultrafiltration tubes and characteristic peptide standards in the present invention to develop an α-synuclein typing detection kit suitable for clinical testing;

[0285] Technical adaptability: Compatibility design based on conventional mass spectrometry platforms (such as triple quadrupole mass spectrometry) can quickly adapt to existing hospital mass spectrometry laboratory equipment, reducing the conversion cost.

[0286] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A synchronous detection method for A53T mutant and wild-type α-synuclein, characterized in that, The synchronous detection method includes the following steps: S1 Preprocess the sample: S2 Detect the preprocessed sample with a liquid chromatography - mass spectrometer to obtain the response signals of the characteristic peptides of A53T mutant and wild - type α - synuclein in the liquid chromatography - mass spectrometer; Among them, the characteristic peptide 1 of A53T mutant α - synuclein is EGVVHGVTTVAEK; The characteristic peptide 2 of wild - type α - synuclein is EGVVHGVATVAEK; S3 Convert the response signals of the characteristic peptides obtained in step S2 in the liquid chromatography - mass spectrometer into the contents of A53T mutant and wild - type α - synuclein in the sample according to the standard curve.

2. The synchronous detection method according to claim 1, wherein The sample preprocessing method in step S1 includes the following steps: I Concentrate and ultrafilter the sample and collect the filtrate; II Add a buffer solution to the filtrate and incubate with shaking; III Add a protein denaturant in step II and incubate; IV Add a protein stabilizer in step III, incubate, and then lyophilize the sample; V Redissolve the lyophilized sample in step IV with a buffer solution, add trypsin for enzymatic digestion, and then lyophilize the sample.

3. The synchronous detection method according to claim 1, characterized in that The mass spectrometry conditions of the liquid chromatography - mass spectrometer in step S2 meet the following conditions: (1) The parent ion of the characteristic peptide 1 is 442.6, and the daughter ion is 347.2; (2) The declustering voltage of the characteristic peptide 1 is 80V, and the collision energy is 24 - 34eV; (3) The parent ion of the characteristic peptide 2 is 648.4, and the daughter ion is 347.4; (4) The declustering voltage of the characteristic peptide 2 is 80V, and the collision energy is 40 - 50eV.

4. The synchronous detection method according to claim 1, characterized in that, The liquid chromatography conditions of the liquid chromatography - mass spectrometer in step S2 meet the following conditions: (1) The mobile phase is: aqueous phase 0.1% formic acid aqueous solution, organic phase 0.1% formic acid acetonitrile solution; (2) The mobile phase gradient is: 0 - 0.50 min, the volume ratio of the aqueous phase to the organic phase is: 95%:5%; 0.50 - 3.00 min, the volume ratio of the aqueous phase to the organic phase is: (60% - 95%):(5% - 40%); 3.00 - 3.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 60%):(40% - 85%); 3.01 - 4.00 min, the volume ratio of the aqueous phase to the organic phase is: 15%:85%; 4.00 - 4.01 min, the volume ratio of the aqueous phase to the organic phase is: (15% - 95%):(5% - 85%); 4.01 - 5.00 min, the volume ratio of the aqueous phase to the organic phase is: 95%:5%.

5. The synchronous detection method according to claim 1, wherein It also includes the step of detecting the total amount of α - synuclein.

6. The synchronous detection method according to claim 5, wherein The step of detecting the total amount of α - synuclein is: a Detect the preprocessed sample with a liquid chromatography - mass spectrometer to obtain the response signals of the characteristic peptides of α - synuclein in the sample in the liquid chromatography - mass spectrometer; Among them, the characteristic peptide 3 of α - synuclein is QGVAEAAGK; or / and, the characteristic peptide 4 of α - synuclein is TVEGAGSIAAATGFVK; b Convert the response signals of the characteristic peptides obtained in step a in the liquid chromatography - mass spectrometer into the content of α - synuclein in the sample according to the standard curve.

7. The synchronous detection method according to claim 6, wherein The mass spectrometry conditions for detecting the total amount of α-synuclein satisfy the following conditions: (1) The parent ion of the characteristic peptide 3 is 415.7, and the daughter ion is 346.2; (2) The declustering voltage of the characteristic peptide 3 is 80V, and the collision energy is 24 - 34eV; (3) The parent ion of the characteristic peptide 4 is 739.9, and the daughter ion is 173.1; (4) The declustering voltage of the characteristic peptide 4 is 80V, and the collision energy is 40 - 50eV.

8. A characteristic peptide segment for detecting A53T mutant and wild-type α-synuclein, characterized in that, The amino acid sequence of the characteristic peptide for detecting the A53T mutant α-synuclein is as shown in SEQ ID NO: 1, and / or, the amino acid sequence of the characteristic peptide for detecting the wild-type α-synuclein is as shown in SEQ ID NO:

2.

9. A characteristic peptide segment for detecting α-synuclein, characterized in that, The amino acid sequence of the characteristic peptide is as shown in any one of SEQ ID NO: 3 - 4.

10. The application of the characteristic peptide according to claim 8 in the synchronous detection method of A53T mutant and wild-type α-synuclein, or, the application of the characteristic peptide according to claim 9 in the detection method of the total content of α-synuclein.

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