CN115078570B - Application of Tau protein 639-bit thiocyanate amino acid modification in detection of Alzheimer's disease by rapid mass spectrometry - Google Patents
Application of Tau protein 639-bit thiocyanate amino acid modification in detection of Alzheimer's disease by rapid mass spectrometry Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 22
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 230000004048 modification Effects 0.000 title claims abstract description 16
- 238000012986 modification Methods 0.000 title claims abstract description 16
- 238000004949 mass spectrometry Methods 0.000 title claims abstract description 13
- -1 thiocyanate amino acid Chemical class 0.000 title claims description 15
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention relates to the technical field of disease diagnosis and rapid detection, in particular to application of Tau protein 639-thiocyanate amino acid modification in detection of Alzheimer's disease by a rapid mass spectrometry. According to the invention, the research on Alzheimer's disease shows that the amino acid modification of Tau protein 639 thiocyanate only occurs in Alzheimer's disease patients, so that a more accurate detection method is provided for diagnosis of Alzheimer's disease, and the method has good practical application value.
Description
Technical Field
The invention relates to the technical field of disease diagnosis and rapid detection, in particular to application of Tau protein 639-thiocyanate amino acid modification in detection of Alzheimer's disease by a rapid mass spectrometry.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Alzheimer's disease is one of the most common types of dementia in the elderly, and one of the most common chronic diseases in the elderly, accounting for about 50-70% of Alzheimer's disease. One survey in 2016 showed that 4000 tens of thousands of people all over the world had developed alzheimer's disease, and this figure is expected to double every 20 years. With increasing age, the prevalence of Alzheimer's disease gradually increases, with an average age of 6.1 years, with a 1-fold increase in prevalence, up to 20% -30% after the age of 85 years. Alzheimer's disease can be classified into three categories, mild, moderate and severe, depending on the degree of cognitive impairment. Alzheimer's disease can be divided into sporadic and familial forms according to the form of the disease.
Alzheimer's Disease (AD) is a degenerative disease of the nervous system that occurs and develops with a potential. Studies have shown that there is a latent development time of about 20 years before the clinical symptoms of alzheimer's disease appear, i.e. alzheimer's patients have been abnormal in their brain nerve cell metabolism about 20 years before they show clinical symptoms. Because of the strong secrecy of the early stage of the disease, clinical diagnosis of the early stage disease is basically impossible. Clinically, diagnosis of Alzheimer's disease is mainly based on mental scale, detection of Abeta protein amount and tau protein phosphorylation degree in cerebrospinal fluid and serological components are used as auxiliary diagnosis, and brain imaging auxiliary diagnosis such as CT, MRI and the like is also used. There is a lack of more and more effective diagnosis of physiological indicators.
Alzheimer's disease takes brain nerve cell atrophy and death as direct causes, and the abnormal microtubule system of nerve cells is an important factor causing nerve cell atrophy and death. The microtubule system is a component of the nerve cell skeleton, and can maintain the morphology of the nerve cell dendrites and axons and ensure the normal operation of the functions of the nerve cell dendrites and axons. Microtubules consist of tubulin and microtubule-associated proteins, tau protein being microtubule-associated protein. Tau protein binding to tubulin in normal brain nerve cells promotes tubulin polymerization to form microtubules and bind thereto. tau protein is able to maintain microtubule stability, reduce the dissociation rate of tubulin molecules, and induce microtubule bundling. Neural cells regulate synaptic connections between nerves and physiological functions of synapses by regulating the aggregation and dissociation of microtubules. In the early stage of Alzheimer's disease, the problem of the neural cell on microtubule regulation is caused by abnormal reasons, so that the neural cell can not normally control the function of axons, and further the neural cell can shrink and die. It is generally believed that transitional phosphorylation of microtubule-associated protein tau results in abnormal polymerization of tubulin, while excessive tau protein free in nerve cells further contributes to nerve cell death.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide the application of Tau protein 639-thiocyanate amino acid modification in detecting Alzheimer's disease by a rapid mass spectrometry method, and provides a more accurate and effective molecular biological diagnosis mode and treatment reference for Alzheimer's disease patients.
In order to achieve the above object, the technical scheme of the present invention is as follows:
in a first aspect of the invention there is provided the use of the amino thiocyanate acid at position 639 of the Tau protein as a biomarker for Alzheimer's disease.
The study of the invention finds that 639 th thiocyanate amino acid modification in Tau protein molecule has extremely obvious statistical difference between Alzheimer's disease and non-Alzheimer's disease patients, and the data shows that the thiocyanate amino acid only appears in Alzheimer's disease.
Specifically, the invention provides application of Tau protein 639-thiocyanate amino acid serving as a biomarker in preparation of diagnostic products for Alzheimer's disease.
The Tau protein 639-bit thiocyanate amino acid modification in the Alzheimer's disease sample and the non-Alzheimer's disease sample has obvious statistical difference and diagnostic significance.
In a specific embodiment, the biomarker comprises: tau protein 639-thiocyanate amino acid;
in a second aspect of the invention, there is provided a kit for the diagnosis of Alzheimer's disease, comprising reagents for specifically detecting the amino acid thiocyanate at position 639 of the Tau protein;
The kit can be used for detecting the amino acid thiocyanate at position 639 of Tau protein;
The invention also provides a diagnosis method of Alzheimer's disease, which comprises the following steps: detecting Tau protein 639-position thiocyanate amino acid of a sample to be detected, and judging occurrence or progress of Alzheimer's disease;
in a third aspect of the invention, there is provided a therapeutic target for Alzheimer's disease, said therapeutic target being the amino thiocyanate at position 639 of the Tau protein;
the invention provides application of Tau protein 639-bit thiocyanate amino acid as an Alzheimer disease treatment target spot in preparation of Alzheimer disease treatment medicines, and medicines such as different forms of proteins or small molecules which can be prepared aiming at the target spot;
The invention also provides a medicine for treating Alzheimer's disease, which contains a component capable of inhibiting the expression of Tau protein 639 thiocyanate amino acid.
The specific embodiment of the invention has the following beneficial effects:
Clinically, diagnosis of Alzheimer's disease is mainly based on mental scale, which makes diagnosis too subjective. While Tau protein transitional phosphorylation and aβ protein theory can aid in diagnosis to some extent, more and more effective biological detection criteria are clinically needed. The physiological state of the patient is used as a final judgment index, so that the method is more effective for the subsequent diagnosis and treatment of the patient. The study on Alzheimer's disease shows that the modification of Tau protein 639-thiocyanate amino acid only occurs in Alzheimer's disease patients, thereby providing a more accurate detection method for diagnosing Alzheimer's disease and providing a potential treatment target for subsequent treatment, and having good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a second-order spectrum of a target peptide fragment;
FIG. 2 is information on detection of thiocyanate amino acids in a sample;
Fig. 3 is a ROC curve, notes: auc=0.833 for ROC curve, critical value is 0.67.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention is further illustrated and described below in connection with specific examples.
Example 1 technical scheme 1: mass spectrometry of samples for Alzheimer's disease.
In the experimental process, 18 samples are adopted, 9 samples are Alzheimer's disease, and 9 samples are non-Alzheimer's control.
The sample tissue was cut into tissue pieces smaller than 1mm 2, RIPA lysate (tissue: lysate=1:100 (1 mg/100 ul)) was added and mixed with shaking and incubated on ice for 30min.
The ice-bathed grinding beads were added to the sample tube.
The sample tube was placed in a grinder and ground 3 times for 30 seconds each.
The beads were removed, sonicated in an ice bath, 3 times for 5 seconds each, and vortexed for 30 seconds.
Centrifuge at 10000g for 10min at 4 ℃.
The supernatant was taken into a new EP tube, 5 volumes of pre-chilled acetone at-40℃was added to the sample tube, and vortexed for 10 seconds.
The protein was precipitated in a refrigerator at-40℃for 1 hour.
Centrifuging at 4deg.C for 10 min at 10000g, and discarding supernatant.
The protein precipitate was washed by adding 80% acetone (water) by the same volume to the precipitate with shaking.
And adding 100mM ammonium bicarbonate solution into the precipitate to dissolve the precipitate, and carrying out low-temperature ultrasonic dissolution.
The BCA method determines the protein concentration of the sample.
Trypsin [ KR|P ] (abbreviated as Trypsin) was added to the sample tube to total sample protein: trypsin=50:1 (μg: μg).
Incubate at 37℃and 700rpm for 12 hours under nitrogen.
The first 1/2 amount of Trypsin was added to the sample and incubated at 37℃for 2 hours at 700rpm under nitrogen.
The digested peptide was desalted using a (WATERS SEP-Pak Vac 1cc (50 mg) C18cartridge desalting column.
The concentration of the desalted peptide fragment was determined using BCA peptide fragment concentration determination kit.
100 Μg of the peptide fragment was sampled and fractionated using Waterse2695,2695 (Separations Module) liquid phase. ① BufferA: 10ml 100 Xstock solution,970ml water, 20ml CAN (acetonitrile); bufferB:10ml 100 Xstock solution, 10ml water, 980mlACN (acetonitrile); 100×stock solution (100 ml): 13.4ml of concentrated ammonia water and 86.6ml of water. ② The chromatograph is operated and the chromatograph line is flushed. ③ Taking 100ul of sample into a residue-free sample bottle, starting sample loading, setting a liquid phase set flow rate of 0.1ml/min, and setting the temperature of a chromatographic column incubator at 50 ℃, and carrying out Wavelength:214nm; bandwidth:12nm. ④ A 2ml flat bottom EP tube was prepared, labeled in sequence and placed in a sample collector to collect the sample components. ⑤ The collected fractions were separated into three groups in time series, the sample fractions of each group were combined, and the samples were dried by vacuum centrifugation.
The sample was dissolved with 0.1% formic acid (water) and centrifuged at 20000g at 4℃for 10 min.
Separation was performed using an EASY-nLC 1200 liquid phase system and DDA mode detection was performed using an on-line Thermo QE-HFX mass spectrometer. Liquid phase separation system parameters, pre-column: 150 μm 3cm C18.9 μm Reprosil-Pur 120; chromatographic column: 150 μm 25cm C18.9 μm Reprosil-Pur 120; flow rate: 600nl/min; mobile phase a:0.1% formic acid (water); mobile phase B:0.1% formic acid 80% acetonitrile (water); elution gradient: 4% -7%B% for 1min,7% -25% b for 94min, 25% -40% b for 16min,40% -100% b for 5min,100% for 4min; sample loading amount: 5 μl. Mass spectrum detection parameters, scan range: 150-2000m/z; data acquisition mode: DDA acquisition mode, top 20 fragment patterns; MS1 resolution (200 m/z): 60,000; MS1 AGC TARGET:3e 6, maxIT:50ms; MS2 resolution (200 m/z): 15,000; MS2 AGC TARGET:5e 4, maxIT:45ms; normalized Collision Energy (NCE): 28%; isolation window: 1.6m/z.
Example 2 data analysis method for Tau protein 639-thiocyanate amino acid.
Analysis of mass spectrometry showed 2% thiocyanate amino acid in tau protein.
The secondary spectrum of peptide C (+24.99) GSLGNIHHKPGGGQVEVK of amino thiocyanate at position 639 (FIG. 1) which contains more than 97% of ion fragments (sub-ions except b1 and y 1) shows that the result has very high credibility.
The core content of this embodiment is: amino thiocyanate 639. The P value was 0.0081 less than 0.01, i.e. the modification had an extremely significant statistical difference between alzheimer's and non-alzheimer's patients, and the data show that the thiocyanate amino acid only appears in alzheimer's disease. (FIG. 2)
DELTAMASS related information. DELTAMASS to the genus protein: p10636 (tau protein); DELTAMASS position: 639; DELTAMASS peptide fragment: CGSLGNIHHKPGGGQVEVK; DELTAMASS molecular weight: 24.99; DELTAMASS score: 0.924 (deltamass scoring means that in mass spectrometry detection, deltamass results of single deltamass measured by mass spectrometry are in a normal distribution form within the allowable range of accuracy and error due to various extremely small effects, and the score is the fitting degree of a normal curve, the score is 0-1, the higher the score is, the more reliable the detection result is, and a secondary map capable of reaching 0.2 is approved).
ROC curves for tau protein of AD group and control group are shown in figure 3.
From the above information, it can be seen that: the thiocyanate amino acid at position 639 of tau protein is important for diagnosis of patients with Alzheimer's disease.
In the above description of the present embodiment, the target peptide is subjected to subsequent experiments after enzymatic hydrolysis of the sample protein by Trypsin [ KR|P ]. Various enzymatic sample protocols, such as Trypsin(semi)[KR|P];Trypsin/p[KR|-]; TrypsinK[K|P];TrypsinR[R|P];ChymoTrypsin[FWYL|P];ArgC[R|P];AspN[-|D]n-term;Clostripain[R|-];CNBr[M|P];Elastase[GVLIA|P];Formic Acid[D|P];GluC[DE |P];GluC bicarb[E|P];Iodosobenzoate[W|-];LysC[K|P];LysC/P[K|-];LysN[-|K]n-term;LysN promisc[-|KASR]n-term;PepsinA[FL|P];Protein endopeptidase[P|-];Staph protease[E|-];Trypsin-CNBr[KRM|P];Trypsin-GluC[DEKR|P], can also be used in mass spectrometry. The amino acid thiocyanate 639 of tau protein can also be detected in the samples treated by these enzymatic hydrolysis or by physical means such as ultrasound. In addition, the modifications set forth in this example can be similarly enriched and detected by immunological interactions with proteins and the like.
Example 3 application to the amino acid thiocyanate at position 639 of tau protein.
A rapid mass spectrometry detection method for tau protein 639 thiocyanate amino acid.
Mass spectral data from example 2 were analyzed using skyline to yield target peptide fragment C (+24.99) GSLGNIHHKPGGGQVEVK chromatographic retention times. The retention time is combined with the target peptide fragment 2-valent and 3-valent parent ions and their corresponding daughter ions to construct a list of ions of PRMs collected for mass spectrometry detection. ( See table 1. List of ions. Each parent ion may correspond to a plurality of 1,2, 3 valent daughter ions. For ease of detection, this example only lists the relatively high abundance daughter ion fragments with no less than 5 amino acid residues )
TABLE 1 parent 2-and 3-valent ions of the target peptide fragment and the corresponding daughter ions
The collected samples were processed as in example 1.
Separation was performed using an EASY-nLC 1200 liquid phase system, detected using an online Thermo QE-HFX mass spectrometer. The parameters of the liquid phase separation system are pre-column: 75 μm.2cm PepMap c18 μm Thermo; chromatographic column: 75 μm 15cm PepMap C18 2 μm Thermo; flow rate: 500nl/min; mobile phase a:0.1% formic acid (water); mobile phase B:0.1% formic acid 80% acetonitrile (water); elution gradient: 6% -25% B for 21min,25% -40% B for 4min,40% -100% B for 3min, and 100% B for 2min; sample loadings were: 3 μl. Mass spectrum detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragment pattern; MS2 resolution (200 m/z): 30,000; MS2 AGC TARGET:2e 5, maxIT:70ms; normalized Collision Energy (NCE): 28%; isolation window: 1.6m/z.
And adjusting mass spectrum parameters to enable the mass spectrum parameters to rapidly acquire and detect samples according to the PRM ion list.
And analyzing the data measured by the mass spectrometer by using skyline, carrying out relative or absolute quantification on target ion fragments by adopting an internal standard or external standard mode, and then carrying out qualitative or quantitative analysis on the sample according to the quantitative result of the ion fragments.
Whether the patient has Alzheimer's disease or is a criterion for diagnosis thereof is determined under qualitative or quantitative precursors of the sample.
Conventional immunoassays or protein interaction protocols can also be used to detect modifications of interest, and the present example is described in detail only in the PRM protocol.
The thiocyanate amino acid at position 639 of tau protein (Uniprot ID: P10636) described in this example can also be used as a therapeutic target for Alzheimer's disease. Different forms of proteins or small molecule drugs which can be prepared aiming at the target point are feasible.
And (3) detection and verification: the test results of 3 healthy samples and 3 clinically diagnosed samples of Alzheimer's disease (samples not belonging to the analysis set of example 1) were identical to the known results when the diagnosis was performed by using 3 healthy samples and 3 clinically diagnosed samples of Alzheimer's disease as subjects.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. Use of a substance modified by the amino acid thiocyanate at position 639 of Tau protein in the detection of the protein in the preparation of a product for rapid mass spectrometry for the detection of alzheimer, said Tau protein having Uniprot ID number P10636.
2. A kit for diagnosing Alzheimer's disease, which is characterized by comprising a reagent for specifically detecting Tau protein thiocyanate amino acid modification;
The kit can detect the amino acid thiocyanate modification at 639 th site of Tau protein, and the Uniprot ID number of the Tau protein is P10636.
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