LC-MS / MS-based TAR DNA binding protein 43 detection method and application

Abstract

The invention belongs to the technical field of biomedical detection, and discloses a TAR DNA binding protein 43 detection method based on LC-MS / MS and application. The detection method comprises the following steps: taking a biological matrix to be detected, and filtering to obtain filtrate; adding an ion enhancer into the obtained filtrate, oscillating and incubating, adding a protein precipitant, and separating precipitated protein; adding trypsin into the precipitated protein, and incubating to obtain a sample containing a characteristic peptide fragment; and detecting the TAR DNA binding protein 43 in the sample by adopting liquid chromatography-triple quadrupole mass spectrometry. According to the method, the TDP-43 is specifically detected and quantified by adopting a liquid chromatography-tandem mass spectrometry technology, so that the excellent effects of efficiently detecting and accurately quantifying the TDP-43 and improving the detection sensitivity and stability are achieved. The method can be applied to the fields of in-vitro diagnosis of neurodegenerative diseases, pathological mechanism research and therapeutic drug development.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a TAR DNA binding protein 43 detection method based on LC-MS / MS and its application. Background Art

[0002] TAR DNA-binding protein 43 (TDP-43) is a multifunctional nucleic acid-binding protein and a key protein involved in the regulation of RNA metabolism. It participates in various biological processes within the cell, including transcription, translation, and pre-mRNA splicing. Abnormal aggregation and phosphorylation of TAR DNA-binding protein 43 are closely associated with various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTLD), and Alzheimer's disease (AD). In these diseases, TDP-43 translocates from the nucleus to the cytoplasm to form insoluble aggregates, leading to neuronal death and disease progression.

[0003] Currently, the detection of TDP-43 in clinical research mainly relies on immunological methods (such as Western blot, ELISA and immunohistochemistry). These methods achieve qualitative or semi-quantitative analysis of the target protein through antigen-antibody reactions, but have significant limitations in practical applications, such as: antibody-dependent interference, TDP-43 is prone to abnormal modification (such as truncation, phosphorylation or ubiquitination), resulting in the masking of antibody binding epitopes and false-negative results; insufficient quantitative accuracy, immunological methods are limited by the linear detection range of antibodies and matrix effects, making it difficult to achieve high-precision absolute quantification in complex biological samples (such as cerebrospinal fluid and plasma).

[0004] Therefore, there is an urgent need to develop a highly sensitive, highly specific and standardized TDP-43 mass spectrometry detection method to meet the clinical needs of early diagnosis, disease course monitoring and treatment evaluation of neurodegenerative diseases. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method and application of detecting TAR DNA binding protein 43 based on liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for detecting TAR DNA binding protein 43 based on LC-MS / MS, comprising the following steps:

[0008] (1) taking a biological matrix to be tested and filtering to obtain a filtrate; adding an ion enhancer to the obtained filtrate, shaking and incubating, and then adding a protein precipitant to separate and precipitate the protein;

[0009] (2) adding trypsin to the obtained precipitated protein and incubating to obtain a sample containing a characteristic peptide; and detecting TAR DNA binding protein 43 in the sample by liquid chromatography-triple quadrupole mass spectrometry;

[0010] The amino acid sequence of the characteristic peptide segment is at least one of TTEQDLK, EYFSTFGEVLMVQVK, FTEYETQVK, and QSQDEPLR.

[0011] The present invention uses liquid chromatography-tandem mass spectrometry to specifically detect and quantify TAR DNA-binding protein 43 (TDP-43). This method includes key technical elements such as sample pretreatment optimization, characteristic peptide screening, and liquid chromatography-triple quadrupole mass spectrometry detection. In particular, trypsin is used to cleave TDP-43 to generate highly stable characteristic peptides. This method achieves the excellent results of efficient detection and precise quantification of TDP-43 while improving detection sensitivity and stability. It can be applied to the in vitro diagnosis of neurodegenerative diseases, pathological mechanism research, and therapeutic drug development.

[0012] As a preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method described in the present invention, the amino acid sequence FTEYETQVK and / or QSQDEPLR is used as a characteristic peptide segment for quantitative detection of TAR DNA-binding protein 43, which has higher accuracy.

[0013] As a preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method of the present invention, the amino acid sequence EYFSTFGEVLMVQVK is used as a characteristic peptide segment for qualitative detection of TAR DNA-binding protein 43, which has high sensitivity.

[0014] As a preferred embodiment of the LC-MS / MS-based TAR DNA binding protein 43 detection method of the present invention, in step (1), the biological matrix includes at least one of serum, cerebrospinal fluid, plasma and exosomes.

[0015] As a preferred embodiment of the LC-MS / MS-based TAR DNA binding protein 43 detection method of the present invention, in step (1), the filtration uses a 50kD-60kD cut-off ultrafiltration tube.

[0016] Preferably, the filtration uses an ultrafiltration tube with a cut-off value of any one or both of 50kD, 51kD, 52kD, 53kD, 54kD, 55kD, 56kD, 57kD, 58kD, 59kD, and 60kD.

[0017] As a preferred embodiment of the LC-MS / MS-based TAR DNA binding protein 43 detection method of the present invention, in step (1), the ion enhancer is ammonium bicarbonate.

[0018] As a preferred embodiment of the LC-MS / MS-based TAR DNA binding protein 43 detection method of the present invention, in step (1), the protein precipitant includes acetonitrile and / or methanol.

[0019] As a preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method of the present invention, in step (1), after the shaking incubation, DTT solution is added, and after incubation at 50°C-70°C for 1h-2h, iodoacetamide is added, and incubated at room temperature in the dark for 30min-1h, the protein precipitant is added to precipitate the protein, the supernatant is obtained by centrifugation, and ammonium bicarbonate is added to suspend the protein.

[0020] Preferably, the incubation temperature is any one of or both of 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, and 70°C; and the incubation time is any one of or both of 1h, 1.5h, and 2h.

[0021] Preferably, the incubation time at room temperature in the dark is within the range of any one or both of 30 min, 35 min, 40 min, 45 min, 50 min, 55 min and 1 h.

[0022] As a preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method of the present invention, in step (2), the liquid chromatography-triple quadrupole mass spectrometry detection liquid phase model is AB SCIEX ExionLC; the chromatographic column is Acqiuty UPLC® BEH C18, with specifications of 2.1×100 mm and 1.7 μm.

[0023] As a further preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method described in the present invention, the mobile phase of the liquid chromatography-triple quadrupole mass spectrometry detection includes an aqueous phase and an organic phase; the aqueous phase is formic acid and water in a volume ratio of 1:1000-1500, and the organic phase is formic acid and acetonitrile in a volume ratio of 1:1000-1500; the flow rate is 0.3 mL / min-0.5 mL / min; and the column temperature is 35°C-45°C.

[0024] Preferably, the volume ratio of formic acid to water is 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:1500, or any one of the range values ​​of both; the volume ratio of formic acid to acetonitrile is 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, or 1:1500. :4500, 1:1500, any one or both of them; the flow rate is 0.3mL / min, 0.35mL / min, 0.4mL / min, 0.45mL / min, 0.5mL / min, any one or both of them; the column temperature is 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 42℃, 43℃, 44℃, 45℃, any one or both of them.

[0025] As a preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method described in the present invention, in step (2), the mass spectrometer model of the liquid chromatography-triple quadrupole mass spectrometry detection is AB SCIEX 7500, the detection method is multiple reaction ion monitoring, positive ion mode scanning; and the ion source is ESI source.

[0026] As a further preferred embodiment of the LC-MS / MS-based TAR DNA-binding protein 43 detection method described in the present invention, the mass spectrometry conditions are: parent ion (Q1) 400Da-900Da, daughter ion (Q3) 500Da-1010Da, curtain gas (CUR) 35psi-45psi, entrance voltage (EP) 5V-15V, collision voltage (CE) 20V-45V, outlet voltage (CXP) 10V-20V, temperature (TEM) 500℃-600℃, nebulizing gas (GS1) 45psi-55psi, auxiliary heating gas (GS2) 45psi-55psi, dwell time (Dwell time) 45msec-55msec, collision gas (CAD) 8-10.

[0027] As a further preferred embodiment, when the characteristic peptide is TTEQDLK, the mass spectrometry conditions are: parent ion (Q1) 400Da-420Da, daughter ion (Q3) 600Da-640Da, curtain gas (CUR) 40psi-45psi, entrance voltage (EP) 8V-10V, collision voltage (CE) 20V-30V, exit voltage (CXP) 10V-15V, temperature (TEM) 500℃-550℃, nebulizer gas (GS1) 45psi-50psi, auxiliary heating gas (GS2) 45psi-50psi, dwell time (Dwell time) 45msec-50msec, collision gas (CAD) 9-10.

[0028] As a further preferred embodiment, when the characteristic peptide segment is EYFSTFGEVLMVQVK, the mass spectrometry conditions are parent ion (Q1) 880Da-900Da, daughter ion (Q3) 1000Da-1010Da, curtain gas (CUR) 40psi-45psi, entrance voltage (EP) 8V-10V, collision voltage (CE) 40V-45V, exit voltage (CXP) 10V-15V, temperature (TEM) 500℃-550℃, nebulizer gas (GS1) 50psi-55psi, auxiliary heating gas (GS2) 50psi-55psi, dwell time (Dwell time) 45msec-50msec, collision gas (CAD) 8-9.

[0029] As a further preferred embodiment, when the characteristic peptide is FTEYETQVK, the mass spectrometry conditions are parent ion (Q1) 550Da-600Da, daughter ion (Q3) 880Da-900Da, curtain gas (CUR) 40psi-45psi, entrance voltage (EP) 8V-10V, collision voltage (CE) 25V-30V, exit voltage (CXP) 10V-15V, temperature (TEM) 500℃-550℃, nebulizer gas (GS1) 50psi-55psi, auxiliary heating gas (GS2) 50psi-55psi, dwell time (Dwell time) 45msec-50msec, collision gas (CAD) 8-9.

[0030] As a further preferred embodiment, when the characteristic peptide segment is QSQDEPLR, the mass spectrometry conditions are: parent ion (Q1) 480Da-490Da, daughter ion (Q3) 500Da-520Da, curtain gas (CUR) 40psi-45psi, entrance voltage (EP) 8V-10V, collision voltage (CE) 20V-25V, exit voltage (CXP) 15V-20V, temperature (TEM) 550℃-600℃, nebulizer gas (GS1) 50psi-55psi, auxiliary heating gas (GS2) 50psi-55psi, dwell time (Dwell time) 45msec-50msec, collision gas (CAD) 8-9.

[0031] In a second aspect, the present invention provides a characteristic peptide segment for detecting TAR DNA binding protein 43, wherein the amino acid sequence of the characteristic peptide segment is at least one of TTEQDLK, EYFSTFGEVLMVQVK, FTEYETQVK, and QSQDEPLR.

[0032] In a third aspect, the present invention provides an application of the characteristic peptide segment described in the second aspect in the detection of TAR DNA binding protein 43.

[0033] As a further preferred embodiment, the characteristic peptide segment with the amino acid sequence of FTEYETQVK and / or QSQDEPLR is used in the quantitative detection of TAR DNA binding protein 43.

[0034] As a further preferred embodiment, the characteristic peptide segment with the amino acid sequence of EYFSTFGEVLMVQVK is used in the qualitative detection of TAR DNA binding protein 43.

[0035] In a fourth aspect, the LC-MS / MS-based TAR DNA binding protein 43 detection method described in the first aspect of the present invention and the characteristic peptide segment described in the second aspect are used in the in vitro diagnosis and / or pathological mechanism research of neurodegenerative diseases for the purpose of non-disease diagnosis or treatment.

[0036] In a fifth aspect, the LC-MS / MS-based TAR DNA binding protein 43 detection method described in the first aspect of the present invention and the characteristic peptide segment described in the second aspect of the present invention are used in the development and / or preparation of drugs for the treatment of neurodegenerative diseases.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention replaces the traditional antigen-antibody reaction with mass spectrometry, effectively avoiding false negative results caused by abnormal modification of TDP-43 (such as truncation and phosphorylation), and achieves absolute quantification of TDP-43 in complex biological samples (cerebrospinal fluid, plasma, serum, exosomes), overcoming the antibody dependence and quantitative defects of immunological methods.

[0039] 2. The present invention screens characteristic peptides that are highly associated with pathological isoforms of TDP-43, optimizes the sample pretreatment process to reduce the risk of protease degradation, and improves the ionization efficiency of trace TDP-43 through matrix interference removal technology, significantly improving the specificity and sensitivity of mass spectrometry detection.

[0040] 3. This invention establishes a standardized TDP-43 detection system by developing standardized mass spectrometry parameters (such as collision energy and dwell time in multiple reaction monitoring mode) and data analysis processes to ensure the repeatability and cross-platform compatibility of detection results.

[0041] 4. The detection method of the present invention can be applied to the in vitro diagnosis, pathological mechanism research and therapeutic drug development of neurodegenerative diseases, including:

[0042] a) Accurate diagnosis and disease stratification

[0043] Quantitative detection of TDP-43 can significantly improve the accuracy of early diagnosis of TDP-43 protein diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD-TDP), and assist in distinguishing Alzheimer's disease (AD) subtypes and differential diagnosis with other neurodegenerative diseases.

[0044] b) Dynamic monitoring and efficacy evaluation

[0045] Continuously tracking changes in TDP-43 concentrations during the course of the disease provides a reliable biomarker endpoint for drug clinical trials, which will help accelerate the development of neuroprotective agents or anti-aggregation therapies.

[0046] c) Clinical transformation and cost optimization

[0047] Reduce reliance on invasive brain tissue biopsies and achieve non-invasive diagnosis through peripheral fluid testing; combined with automated processes, significantly reduce the cost and operation time of single sample testing, which is conducive to promoting the clinical popularization of TDP-43 quantification technology.

[0048] d) Standardization and cross-platform verification

[0049] Providing internationally accepted TDP-43 mass spectrometry quantitative standards and detection solutions can solve the problem of incomparable experimental results caused by differences in antibody batches in existing immunological methods, laying a technical foundation for multi-center research and regulatory approval. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the chromatogram of the detection linear range;

[0051] Figure 2 This is the standard curve of the characteristic peptide TTEQDLK (417.7→632.3);

[0052] Figure 3 This is the standard curve of the characteristic peptide EYFSTFGEVLMVQVK (888.9→1002.6);

[0053] Figure 4 This is the standard curve of the characteristic peptide FTEYETQVK (572.8→896.4);

[0054] Figure 5 This is the standard curve of the characteristic peptide QSQDEPLR (486.7→514.3). DETAILED DESCRIPTION

[0055] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0057] Example 1: A method for detecting TAR DNA binding protein 43 based on LC-MS / MS

[0058] 1. Reagent preparation

[0059] TDP-43 protein working solution: Take 50 μg of TDP-43 protein and dissolve it in 500 μL of ultrapure water to 0.1 mg / mL as the stock solution. Aliquot and store at -20°C.

[0060] Mobile phase A: Formic acid: Water = 1:1000 by volume. Prepare according to this ratio when using.

[0061] Mobile phase B: Formic acid: Acetonitrile = 1:1000 by volume. Prepare according to this ratio when using.

[0062] Diluent: Take 180 mg of BSA in acetonitrile, add 3 mL of pure water and mix well. Prepare according to this ratio when using.

[0063] Standard curve and quality control samples: Dilute the TDP-43 protein working solution with 6% BSA. Specific dilution methods are shown in Tables 1 and 2. Prepare according to the proportions before use.

[0064] Table 1 Standard curve sample preparation table

[0065] pipe number Standard concentration (ng / mL) Standard volume (μL) Matrix volume (μL) Final concentration (ng / mL) STD7 100000 100 1900 5000 STD6 5000 1200 800 3000 STD5 3000 1200 600 2000 STD4 2000 1000 1000 1000 STD3 1000 1000 1000 500 STD2 500 1000 1500 200 STD1 200 1000 4000 40

[0066] Table 2 Quality control sample preparation table

[0067]

[0068] 2. Sample pretreatment and standard curve linear regression

[0069] 150 μL of the standard curve samples and quality control samples prepared with blank matrix at different concentrations were taken respectively and centrifuged in a 50 kD cut-off ultrafiltration tube. The filtrate in the outer tube was collected and added with 100 μL of 100 mM ammonium bicarbonate. After incubation at 90°C with shaking for 25 min, 20 μL of 10 mM DTT solution was added. After incubation at 60°C for 1 h, 10 μL of 100 mM iodoacetamide was added. The mixture was incubated at room temperature in the dark for 30 min. Acetonitrile was added to precipitate the protein. The mixture was centrifuged at 10,000 g / min for 10 min. The supernatant was discarded and 100 μL of 50 mM ammonium bicarbonate was added to suspend the protein. 30 μL of 0.2 μg / μL trypsin was added and mixed. The mixture was incubated at 37°C overnight. The sample was lyophilized and then reconstituted with 150 μL of 0.1% formic acid aqueous solution. The sample was centrifuged at 10,000 g / min for 10 min and the supernatant was injected.

[0070] Record the chromatographic peak area of ​​the characteristic peptide in the standard curve sample, and perform linear regression with the peak area y corresponding to its concentration x to obtain the regression equation of the standard curve with a weight coefficient of 1 / x 2 .

[0071] 3. Linear range of detection

[0072] A series of blood samples for the standard curve (see STD1 to STD7 in Example 1) were pretreated and injected. The mass spectrometry conditions were as follows:

[0073] Liquid phase model: AB SCIEX ExionLC; chromatographic column: Acqiuty UPLC® BEH C18 (2.1×100mm, 1.7µm).

[0074] Mobile phase: A (aqueous phase): formic acid: water (1:1000), B (organic phase): formic acid: acetonitrile (1:1000). Flow rate: 0.3 mL / min, column temperature: 40°C. Gradient elution mode: see Table 3.

[0075] Table 3 Gradient elution method

[0076] Time(min) Flow rate (mL / min) A(％) B(％) 0 0.3 80 20 2.00 0.3 60 40 3.00 0.3 40 60 3.01 0.3 0 100 4.00 0.3 0 100 4.01 0.3 80 20 5.00 0.3 80 20

[0077] Mass spectrometer model: AB SCIEX 7500, detection method: multiple reaction monitoring (MRM), positive ion mode scanning; ion source: ESI source, specific mass spectrometer parameters are shown in Table 4.

[0078] Table 4 Mass spectrometry parameters

[0079] parameter TTEQDLK EYFSTFGEVLMVQVK FTEYETQVK QSQDEPLR Precursor ion Q1 (Da) 417.7 888.9 572.8 486.7 Product ion Q3 (Da) 632.3 1002.6 896.4 514.3 Curtain air CUR (psi) 40 40 40 40 Input voltage EP(V) 10 10 10 10 Crash voltage CE(V) 20 43 27 25 Output voltage CXP(V) 15 15 15 15 Temperature TEM (℃) 550 550 550 550 Atomizing gas GS1 (psi) 50 50 50 50 Auxiliary heating gas GS2 (psi) 50 50 50 50 Dwell time (msec) 50 50 50 50 Collision Gas CAD 9 9 9 9

[0080] Record the characteristic peptide chromatographic peak area, perform linear regression with the peak area y corresponding to its concentration x, and obtain the regression equation of the standard curve with a weight coefficient of 1 / x 2 , r>0.99, the results are acceptable. The test results are shown in Table 5 and Figure 1 .

[0081] Table 5 Linear range test results

[0082]

[0083] The above test results show that the use of characteristic peptides FTEYETQVK and QSQDEPLR for quantitative detection of TAR DNA binding protein 43 has higher accuracy; the use of characteristic peptide EYFSTFGEVLMVQVK for qualitative detection of TAR DNA binding protein 43 has higher sensitivity.

[0084] Example 2: Evaluation of detection methods

[0085] The precision, accuracy, and stability of the detection method of Example 1 were evaluated as follows:

[0086] 1. Precision and accuracy of detection

[0087] Quality control blood samples (LQC, MQC, and HQC) were pretreated and injected. Three replicates per batch and concentration were tested over at least two days. The mean intra- and inter-batch accuracy values ​​were within ±15% of the labeled value, and the intra- and inter-batch precision %CVs were within 15%. The test results are shown in Tables 6 and 7.

[0088] Table 6 Test results of precision and accuracy (FTEYETQVK)

[0089]

[0090]

[0091] Table 7 Test results of precision and accuracy (QSQDEPLR)

[0092]

[0093] 2. Detection stability of long-term frozen samples

[0094] Three quality control samples (LQC, MQC, and HQC) were prepared from blank matrix and stored at -80°C for 15 days. After pretreatment, the samples were injected. The peak areas of the characteristic peptides were recorded, and the protein concentrations were calculated from the standard curve for the day. When compared with the labeled concentration, the average accuracy was within ±15% of the labeled concentration. The test results are shown in Table 8.

[0095] Table 8 Test results of samples frozen for 15 days

[0096]

[0097] 3. Detection stability of samples stored at room temperature

[0098] Three quality control samples (LQC, MQC, and HQC) were prepared using blank matrix and stored at room temperature for 8 hours. After pretreatment, they were injected. The peak areas of the characteristic peptides were recorded, and the protein concentrations were calculated from the standard curve for the day. When compared with the labeled concentration, the average accuracy was within ±15% of the labeled concentration. The test results are shown in Table 9.

[0099] Table 9 Test results of samples at room temperature for 8 hours

[0100]

[0101]

[0102] 4. Detection stability of samples subjected to repeated freeze-thaw cycles

[0103] Three quality control samples (LQC, MQC, and HQC) were prepared using blank matrix. These samples were frozen and thawed three times at -80°C (each freeze-thaw cycle lasting more than 12 hours). After pretreatment, the samples were injected. The peak areas of the characteristic peptides were recorded, and the protein concentrations were calculated from the standard curve for the day. When compared with the labeled concentration, the average accuracy was within ±15% of the labeled concentration. The test results are shown in Table 10.

[0104] Table 10 Test results of repeated freeze-thaw samples

[0105]

[0106]

[0107] 5. Detection stability of repeated sample injections

[0108] Three quality control samples (LQC, MQC, and HQC) were prepared using blank matrix and injected three times after pretreatment. The peak areas of the characteristic peptides were recorded, and the protein concentrations were calculated from the standard curve for the day. When compared with the labeled concentration, the average accuracy was within ±15% of the labeled concentration. The test results are shown in Table 11.

[0109] Table 11 Test results of repeated injection samples

[0110]

[0111]

[0112] In summary, the LC-MS / MS-based TAR DNA binding protein 43 detection method of the present invention has the following advantages:

[0113] (1) High specificity and sensitivity

[0114] Targeted proteomics technology based on mass spectrometry, through specific peptide selection and multiple reaction monitoring (PRM / MRM), has a detection limit as low as nanograms per milliliter, and can accurately quantify TDP-43 in trace samples (such as cerebrospinal fluid and exosomes), significantly superior to traditional ELISA or Western blot methods.

[0115] (2) Absolute quantification and standardization

[0116] It can achieve absolute quantification of TDP-43, overcoming the batch-to-batch variability and antibody titer dependence of traditional semi-quantitative techniques (such as immunoblotting), improving data comparability, and achieving consistency with clinical diagnosis.

[0117] (3) Strong anti-interference ability and sample universality

[0118] The detection method of the present invention has high tolerance and specificity to sample matrix effects (such as high salt in cerebrospinal fluid and lipids in blood), can directly analyze complex biological samples without complex pretreatment, and is suitable for TDP-43 detection in various sources such as brain tissue, peripheral body fluids and cell models.

[0119] (4) Technical compatibility and scalability

[0120] It can seamlessly connect the automated pre-processing platform with the high-throughput mass spectrometry system to meet the needs of large-scale clinical screening. By adjusting the characteristic peptide library, it can be quickly expanded to the detection of new mutants or modified types of TDP-43, and can be applied to the in vitro diagnosis of neurodegenerative diseases, pathological mechanism research, and therapeutic drug development.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting TAR DNA binding protein 43 based on LC-MS / MS, characterized in that: The following steps are involved: (1) taking a biological matrix to be tested and filtering to obtain a filtrate; adding an ion enhancer to the obtained filtrate, shaking and incubating, and then adding a protein precipitant to separate and precipitate the protein; (2) adding trypsin to the obtained precipitated protein and incubating to obtain a sample containing a characteristic peptide; and detecting TAR DNA binding protein 43 in the sample by liquid chromatography-triple quadrupole mass spectrometry; The amino acid sequence of the characteristic peptide segment is at least one of TTEQDLK, EYFSTFGEVLMVQVK, FTEYETQVK, and QSQDEPLR.

2. The LC-MS / MS-based TAR DNA binding protein 43 detection method according to claim 1, characterized in that: In step (1), the biological matrix includes at least one of serum, cerebrospinal fluid, plasma, and exosomes; And / or, the filtration uses a 50kD-60kD cut-off ultrafiltration tube; and / or, the ion enhancer is ammonium bicarbonate; And / or, the protein precipitant comprises acetonitrile and / or methanol.

3. The LC-MS / MS-based TAR DNA binding protein 43 detection method according to claim 1, characterized in that: In step (1), after the shaking incubation, DTT solution is added, and after incubation at 50°C-70°C for 1h-2h, iodoacetamide is added, and incubated at room temperature in the dark for 30min-1h, the protein precipitant is added to precipitate the protein, the supernatant is collected by centrifugation, and ammonium bicarbonate is added to suspend the protein.

4. The method for detecting TAR DNA binding protein 43 based on LC-MS / MS according to claim 1, characterized in that: In step (2), the liquid chromatography-triple quadrupole mass spectrometry detection liquid phase model is AB SCIEX ExionLC; the chromatographic column is Acqiuty UPLC®BEH C18, with specifications of 2.1×100 mm and 1.7 μm.

5. The LC-MS / MS-based TAR DNA binding protein 43 detection method according to claim 4, characterized in that: The mobile phase of the liquid chromatography-triple quadrupole mass spectrometry detection includes an aqueous phase and an organic phase; the aqueous phase is formic acid and water in a volume ratio of 1:1000-1500, and the organic phase is formic acid and acetonitrile in a volume ratio of 1:1000-1500; And / or, the flow rate is 0.3 mL / min-0.5 mL / min; the column temperature is 35° C.-45° C.

6. The method for detecting TAR DNA binding protein 43 based on LC-MS / MS according to claim 1, characterized in that: In step (2), the mass spectrometer model of the liquid chromatography-triple quadrupole mass spectrometry detection is AB SCIEX 7500, the detection method is multiple reaction ion monitoring, positive ion mode scanning; and the ion source is ESI source.

7. The LC-MS / MS-based TAR DNA binding protein 43 detection method according to claim 6, characterized in that: The mass spectrometry conditions are as follows: parent ion 400Da-900 Da, daughter ion 500Da-1010Da, curtain gas 35psi-45psi, inlet voltage 5V-15V, collision voltage 20V-45V, outlet voltage 10V-20V, temperature 500℃-600℃, nebulizer gas 45psi-55psi, auxiliary heating gas 45psi-55psi, dwell time 45msec-55msec, collision gas 8-10.

8. A characteristic peptide segment for detecting TAR DNA binding protein 43, characterized in that: The amino acid sequence of the characteristic peptide segment is at least one of TTEQDLK, EYFSTFGEVLMVQVK, FTEYETQVK, and QSQDEPLR.

9. Use of the characteristic peptide segment according to claim 8 in a method for detecting TAR DNA binding protein 43.

10. Use of the LC-MS / MS-based TAR DNA-binding protein 43 detection method according to any one of claims 1 to 7 and the characteristic peptide segment according to claim 8 in in vitro diagnosis and / or pathological mechanism research of neurodegenerative diseases for purposes other than diagnosis or treatment of diseases.

11. Use of the LC-MS / MS-based TAR DNA binding protein 43 detection method according to any one of claims 1 to 7 and the characteristic peptide segment according to claim 8 in the development and / or preparation of drugs for the treatment of neurodegenerative diseases.

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