CN114487413A - Application of serum polypeptide diagnostic marker Ezrin of brain glioma - Google Patents
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Abstract
The invention discloses a serum polypeptide molecule of a patient with brain glioma and application thereof, wherein the amino acid sequence of a serum polypeptide diagnostic marker Ezrin is shown as SEQ.ID.NO.1, Ezrin is human Ezrin, and the accurate molecular weight of the Ezrin is 1947.02 daltons. The Ezrin shows specific high expression in the serum detection of the patient with the brain glioma, and the expression level of the Ezrin is detected by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an ELISA method, so that the Ezrin can be used as the serum detection method of the patient with the brain glioma.
Description
Technical Field
The invention belongs to the technical field of biomarkers, and particularly relates to a serum polypeptide diagnostic marker Ezrin of brain glioma, and a detection method and application thereof.
Background
Brain gliomas (gliomas) are the most common primary tumors in the central nervous system, accounting for approximately 50% of all central nervous system tumors. Brain gliomas are classified into astrocytomas, oligodendrogliomas, medulloblastomas, ependymomas, and the like according to their cell morphology. Currently, the diagnosis and classification of brain gliomas are mainly based on histopathology and imaging, both of which have their own limitations. The World Health Organization (WHO) divides brain glioma into four grades I-IV, wherein the grades I and II are low-grade glioma, the grades III and IV are high-grade glioma, wherein the high-grade glioma is more invasive, and the prognosis of a patient is worse. To date, there is no effective way to cure the disease, and surgical resection and early-stage chemoradiotherapy can prolong the life of patients.
The following biomarkers are the main biomarkers for brain glioma research in recent years: glial Fibrillary Acidic Protein (GFAP), hyaluronic acid binding protein (BEHAB), matrix metalloproteinase-9 (MMP-9), Human Cytomegalovirus (HCMV), fatty acid binding protein (FABP 7), and the like. Although the research on brain glioma has made a major breakthrough in all aspects, the biomarker lacks sensitivity and specificity for early screening and diagnosis of brain glioma, and a very ideal one in all aspects of diagnosis, treatment, prognosis and monitoring has not been found yet. Therefore, the accurate molecular mechanism of occurrence and development of the brain glioma is discussed, and the new specific diagnosis marker is searched, so that the clinical significance for early diagnosis and treatment of the brain glioma and improvement of the life quality of patients is achieved.
Before any pathological change occurs in any disease, the intracellular proteins are altered in composition and quantity and are reflected by the pattern of proteins in the serum. Therefore, by comparing the expression of different proteins in the serum of different disease populations, it is possible to screen out disease-related marker molecules. Serum proteomics refers to the study of all proteins expressed in the serum of a selected target population, and on the basis of establishing a normal Protein Expression Map (PEM), differential protein spots of the proteins are searched, and disease-related proteins are identified, so that the structure and function of the proteins are further studied, and a new way is developed for studying major disease pathophysiological mechanisms, specific markers for early diagnosis, drug action targets and the like. A large amount of proteins and polypeptides exist in human serum, and the existence, deletion and expression of partial proteins and polypeptides are closely related to the health degree of human beings, so that the human serum becomes a biomarker for disease diagnosis.
Serum diagnosis is considered to be the most recent and effective method for early diagnosis of cancer. The method judges the occurrence and development of the tumor by searching a tumor marker in blood, particularly a protein marker in the blood, so as to realize early diagnosis of the tumor. A large amount of proteins and polypeptides exist in human serum, and the existence, deletion and expression of partial proteins and polypeptides are closely related to the health degree of human beings, so that the human serum becomes a biomarker for disease diagnosis.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a serum polypeptide molecule for diagnosing brain glioma, a detection method and application thereof, wherein the molecular polypeptide is human Ezrin (Ezrin), and is a serum marker of brain glioma.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses an application of a serum polypeptide molecular diagnostic marker Ezrin in the preparation of a brain glioma serum diagnostic reagent, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker Ezrin is shown in SEQ ID No. 1.
Preferably, the molecular weight of the serum polypeptide molecular diagnostic marker Ezrin is 1947.02 daltons.
Preferably, the detection parameter of the serum polypeptide molecular diagnostic marker Ezrin in the serum of a patient with brain glioma is 55.035-127.55pg/mL, and the detection parameter in the serum of a normal healthy population is 43.81-96.255 pg/mL.
Preferably, the serum polypeptide molecular diagnostic marker Ezrin is specifically and highly expressed in the serum sample of the brain glioma patient compared with the serum sample of the normal healthy population.
Preferably, the brain glioma serum diagnostic marker is a serum polypeptide molecule for detecting brain glioma by ELISA.
Further preferably, the serum polypeptide molecular diagnostic marker Ezrin is a novel target point for ELISA detection marker.
The invention also discloses application of a molecule combined with the serum polypeptide molecular diagnosis marker Ezrin in preparing a serum diagnosis reagent for the brain glioma, wherein the amino acid sequence of the serum polypeptide molecular diagnosis marker Ezrin is shown in SEQ ID No. 1.
The invention also discloses a diagnostic kit for brain glioma, which comprises a serum polypeptide molecular diagnostic marker Ezrin, wherein the amino acid sequence of the Ezrin is shown in SEQ ID No. 1.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a serum polypeptide molecule of brain glioma, the amino acid sequence of which is shown in SEQ.ID.NO.1, and the molecule is called Ezrin. Ezrin is human Ezrin, with an exact molecular weight of 1947.02 daltons. Ezrin exhibits specifically high expression in serum detection of brain glioma patients: the range of expression in serum in normal healthy people is: 43.81-96.255 pg/mL; the expression range in the serum of patients with brain glioma is as follows: 55.035-127.55pg/mL, and there was a very significant difference in expression between the two groups (p < 0.01).
In view of the specific high expression of Ezrin in the serum of the brain glioma, Ezrin can be used as a diagnostic marker of the serum of the brain glioma; the expression level of Ezrin is detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ELISA, and can be used as a method for detecting patients with brain glioma. And for ELISA detection of glioma serum diagnosis, Ezrin can be used as a new target point of ELISA detection drugs.
Drawings
FIG. 1 shows the protein polypeptide peaks m/z of the present invention: 1947.02 differential protein polypeptide expression profiles in brain glioma patients (red) and in normal healthy population groups (green);
FIG. 2 is a diagram of gel chromatographic separation of a serum protein polypeptide mixture of a patient with brain glioma; wherein, the abscissa in the chromatogram represents the sample outflow time, and the ordinate represents the relative abundance of the polypeptide;
FIG. 3 is a MS/MS mass spectrometric identification profile of Ezrin of the present invention; wherein, m/z: 1947.02, z is 3;
FIG. 4 shows the expression level of Ezrin protein in the serum of patients and normal healthy persons according to the present invention; wherein, the glioma group N is 30, and the normal healthy population N is 25.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The brain glioma serum polypeptide molecule provided by the invention is a newly screened brain glioma serum diagnostic marker, has specificity in expression, and can be applied to diagnosis of brain glioma. The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
The screening method of the brain glioma serum diagnosis marker specifically comprises the following steps:
the method comprises the steps of firstly, separating and extracting serum protein polypeptides of a brain glioma patient and a normal healthy population by using a liquid protein chip technology, capturing serum protein polypeptide spectrograms of the brain glioma patient and the normal healthy population by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, comparing and analyzing differences of the serum protein polypeptide spectrogram of the brain glioma patient and the serum protein polypeptide spectrogram of the normal healthy population by using ClinProTools2.1 software, finding out protein polypeptide molecules with significant differential expression among groups, and screening out a brain glioma serum marker in a protein polypeptide peak with significant low expression in the serum of the brain glioma patient.
The screened brain glioma polypeptide serum diagnosis markers are verified as follows:
a protein polypeptide mixture separated from the serum of a patient with brain glioma is divided into 20-30 components by using High Performance Liquid Chromatography (HPLC), secondary mass spectrum identification is performed on the protein polypeptide mixture, an enzyme-linked immunosorbent assay is performed on the identified protein polypeptide, and the result of serum regression verification proves that the protein polypeptide mixture is remarkably high-expressed in the serum of the patient with glioma, has specificity, and can be used as a biomarker for screening the serum of the patient with glioma.
1. Sample collection and processing
Samples were collected from 55 (22 men; 33 women) patients with brain glioma, 48 normal healthy people (18 men; 30 women) at Yanyang hospital neurosurgery (9-2021, 3, 2020, Yanan university). The sample considers factors such as age, sex, collection time, whether the storage conditions are consistent, whether basic diseases exist and the like. Collecting blood of the collected person with fasting state, collecting 5mL whole blood with vacuum blood collection tube (yellow cap, with isolation gel), and standing at room temperature for 30 min; centrifuging at room temperature for 5min (3000g), subpackaging the upper layer serum into 100 μ L/tube, immediately storing at-80 deg.C, and avoiding repeated freeze thawing.
Reagents and instrumentation:
the serum proteins were extracted using the magnetic bead kit "weak cationic" (MB-WCX) from Bruker, Germany, as well as spectrally pure (HPLC grade) acetonitrile, trifluoroacetic acid (Merck, Germany) and alpha-cyano-4-hydroxycinnamic acid (HCCA) (Sigma, USA).
The instrument used for the experiment included a magnetic bead separator, 600/384AnchorChip target plate, and an AutoFlexIII Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS) (Bruker Daltonics, Germany).
2. Preparation of serum protein samples
A weak cation (MB-WCX) magnetic bead kit is used for capturing serum protein polypeptide, and the specific operation steps are as follows:
completely mixing the magnetic bead suspension for 1min by using a mixer;
adding 10 mu L of MB-WCX binding solution and 10 mu L of MB-WCX magnetic beads into a PCR tube, uniformly mixing, adding 5 mu L of serum, uniformly mixing for at least 5 times, and standing for 5 min;
thirdly, placing the PCR tube into a magnetic column separator, making the magnetic beads adhere to the wall for 1min, and removing the supernatant after the liquid is clear;
adding 100 mu LMB-WCX flushing fluid, moving the PCR tube on a magnetic column separator back and forth for 10 times, removing supernatant after the magnetic beads are attached to the wall, and repeating the steps three and four times;
adding 5 mu LMB-WCX eluent to wash adherent magnetic beads, repeatedly blowing and beating for 10 times, allowing the magnetic beads to adhere for 2min, and transferring the supernatant into a clean centrifuge tube;
sixthly, adding 5 mu of LMB-WCX stable solution into a centrifuge tube and mixing uniformly, wherein the extracted protein polypeptide can be used for direct MALDI-TOF-MS detection or frozen in a refrigerator at the temperature of-20 ℃ for mass spectrometry within 24 hours.
Mass spectrometry analysis:
mixing 1 μ L of the separated and collected protein sample with 10 μ L of matrix alpha-cyano-4-hydroxycinnamic acid, and spotting 1 μ L of the mixture on an Anchorchip target plate (Bruker, Germany), wherein each sample is spotted with three targets for three times. And after drying at room temperature, putting the target plate into a mass spectrometer for flight time mass spectrometry, correcting the standard substance by adopting FlexControl 2.0 software, and then starting sample detection, wherein each sample generates a mass spectrogram after being subjected to laser targeting for 300 times (5 times of point targeting and 2 times of 30 times of targeting each time), so as to obtain protein polypeptide spectrograms consisting of different mass-to-nuclear ratios (m/z). The protein polypeptide maps of the two groups of serum samples are analyzed by using ClinProTools2.1 software, a genetic algorithm and other biological statistics and bioinformatics methods. Carrying out normalization smoothing treatment on the total ion flow diagram, and eliminating chemical and electro-physical noises; analyzing the difference protein among groups, calculating the difference size, and arranging the difference size from large to small to find out the protein polypeptide peak value (P <0.001) with obvious difference in expression among groups.
After the serum samples of the patient with the brain glioma and the normal healthy population are processed by a magnetic bead separation system and are analyzed by MALDI-TOF-MS, protein polypeptide maps of each sample of the patient with the brain glioma and the normal healthy population are drawn, 143 protein polypeptide peak maps are detected in a molecular weight range of 800 Da-10000 Da, and the three-time repeated stability of each sample is high.
ClinProTools2.1 software is adopted to analyze serum protein polypeptide spectra of brain glioma patients and normal healthy people captured by mass spectrometry, the serum polypeptide spectra of the brain glioma patients and the normal healthy people are compared and analyzed, a protein polypeptide peak image (P <0.001) with extremely obvious difference is detected, and protein polypeptides with obvious high expression in the brain glioma patients are analyzed, and the specific table is shown in Table 1.
TABLE 1 Mass Spectrometry results of serum protein polypeptide profiling of patients with brain glioma and normal healthy population
Molecular weight (mass to charge ratio) | P value | Mean expression of brain gliomas | Average expression of normal healthy population |
1947.02↑ | <0.000001 | 25.46±17.2 | 11.91±8.55 |
The results of Flex analysis software analysis of specific high expression protein polypeptides in the serum of the patient with glioma in Table 1 are shown in FIG. 1, and are obtained by m/z: 1947.02 in brain glioma patients (red) compared to normal healthy population (green), m/z: 1947.02 is highly expressed in the serum of patients with glioma, so it is first identified by sequence identification and used as a marker.
3. Sequence identification of potential brain glioma serum markers
Specifically, the method adopts the technology of combining liquid chromatography separation and mass spectrometry to carry out the treatment on the serum polypeptide marker m/z of a patient with glioma: 1947.02, performing two-dimensional gel chromatography on the serum protein polypeptide remained after the mass spectrum is loaded and collected by magnetic bead separation by adopting a nano-liter flow rate HPLC liquid phase system Easy nLC 1200 of Thermo Scientific company, and performing two-dimensional gel chromatography on the protein polypeptide m/z with the expression up-regulated in the serum of a patient with brain glioma by combining an Orbitrap Fusion mass spectrometer of Thermo Scientific company: 1947.02 for sequence identification.
The specific operation steps are as follows:
3.1 sample Pre-treatment
mu.L of the sample was mixed with 800. mu.L of 5% acetonitrile 0.5% formic acid solution and added to 1.5ml of a less than 10kDa ultrafiltration tube, centrifuged at 7000 rpm for 40min in a cryocentrifuge set at 20 ℃ and concentrated to 500. mu.L. Desalting, eluting polypeptide with 70% methanol and 0.5% formic acid, collecting and concentrating to 100 μ L, and treating with Zip-tip column for concentration.
3.2 chromatographic separation
Each sample was separated using a nanoliter flow rate HPLC liquid system Easy nLC 1200. Buffer solution: the solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile solution. The column was equilibrated with 95% of solution A. The sample was applied to a mass spectrometric pre-column C18 trap column (C183 m 0.10.10X 20mm) and separated by an analytical column C18 column (C181.9m 0.15X 120mm) at a flow rate of 600 nl/min. The relevant liquid phase gradients are shown in table 2 and the results of gel chromatography are shown in figure 2.
TABLE 2 associated liquid phase gradients
3.3 Mass Spectrometry identification
Each sample was separated by capillary HPLC and subjected to mass spectrometry using an Orbitrap Fusion mass spectrometer (Thermo scientific). The main parameter settings are shown in table 3, and the mass spectrometric identification results are shown in fig. 3.
Table 3 main parameter settings for mass spectrometry
3.4 data analysis
Mass spectrometry analysis of RAW data into RAW files, using software sequent and protocol discover 2.5.0(Thermo Scientific) to perform library search identification in a database "uniprot-human", submitting the RAW files to a sequent server through a protocol discover when searching libraries, selecting the well-established database, and then performing database search, wherein relevant parameters are shown in table 4:
TABLE 4 relevant parameters for database search
The result filtering parameters were: peptide FDR is less than or equal to 0.01.
The search results are shown in table 5:
TABLE 5 search results of database search
Therefore, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used for detecting Ezrin or an ELISA method is used for detecting the expression level of Ezrin, and the method can be used for detecting patients with brain glioma. Suggesting that Ezrin is a protein specifically associated with brain glioma and further validated by ELISA assay.
4. ELISA serum verification analysis of brain glioma serum Ezrin expression
4.1 serum samples
Serum verification analysis by ELISA was performed by collecting 30 cases of serum from patients with brain glioma (16 cases for men; 14 cases for women) and 25 cases of serum from normal healthy population (13 cases for men; 12 cases for women). All serum samples were obtained from Yan Yang Hospital, Yanan university, and collected from 9 months to 3 months in 2021 year 2020.
4.2 detection method
The expression level of the serum Ezrin of a patient with brain glioma and a normal healthy population is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the kit is purchased from China Hengyuan biological company. The kit is used for determining the level of human Ezrin (Ezrin) in a specimen by using a double-antibody sandwich method. Coating a microporous plate with a purified human Ezrin antibody to prepare a solid-phase antibody, sequentially adding Ezrin to the micropores coated with the monoclonal antibody, combining with an HRP-labeled Ezrin antibody to form an antibody-antigen-enzyme-labeled antibody compound, and adding a substrate TMB for color development after thorough washing. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The shade of the color was positively correlated with the amount of Ezrin (Ezrin) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of human Ezrin (Ezrin) in the sample was calculated from the standard curve. The specific experimental steps refer to the kit specification, and the positive judgment standard is defined according to the kit specification.
4.3 statistical methods
The independent sample T test was performed using SPSS20 software.
4.4 analysis of results
The results of the analysis by the enzyme linked immunosorbent assay show that the expression level of Ezrin in different detection groups is greater than that of normal healthy patients of brain glioma patients, and the two groups have significant differences, and the specific results are shown in table 6 and fig. 4:
TABLE 6 expression levels of Ezrin protein in sera of different groups
ELISA detection is carried out on Ezrin in serum of patients with brain glioma and normal healthy people, and the result shows that the expression of Ezrin has specificity: the range of expression in serum in brain glioma patients was: 55.035-127.55 pg/mL; the range of expression in serum in normal healthy people is: 43.81-96.255pg/mL, and there was a very significant difference in expression between the two groups (p < 0.01). This indicates that: ezrin is a closely related protein of a patient with brain glioma and can be used as a primary detection index of the brain glioma lesion.
Therefore, the Ezrin expression of the serum sample to be detected can be preliminarily judged to be a patient with brain glioma (55.035-127.55pg/mL) or a normal healthy population (43.81-96.255pg/mL) through an ELISA experiment.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<110> university of west ampere traffic
<120> application of serum polypeptide diagnostic marker Ezrin for brain glioma
<141> 2021-11-25
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<170> SIPOSequenceListing 1.0
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<212> PRT
<213> Artificial Sequence
<400> 1
Lys Ile Ala Gln Asp Leu Glu Met Tyr Gly Ile Asn Tyr Phe Glu Ile
1 5 10 15
Lys Asn
Claims (8)
1. The application of the serum polypeptide molecular diagnostic marker Ezrin in the preparation of a brain glioma serum diagnostic reagent is characterized in that the amino acid sequence of the serum polypeptide molecular diagnostic marker Ezrin is shown in SEQ ID No. 1.
2. The use of claim 1, wherein the molecular weight of Ezrin, a serum polypeptide molecular diagnostic marker, is 1947.02 daltons.
3. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker Ezrin is detected in serum of patients with brain glioma at 55.035-127.55pg/mL and in serum of normal healthy population at 43.81-96.255 pg/mL.
4. The use according to claim 1, wherein the serum polypeptide molecular diagnostic marker Ezrin is specifically highly expressed in serum samples from patients with brain glioma, compared to serum samples from normal healthy populations.
5. The use according to claim 1, wherein the serum diagnostic marker for brain glioma is a serum polypeptide molecule for detecting brain glioma by ELISA.
6. The use according to claim 5, wherein the serum polypeptide molecular diagnostic marker Ezrin is a novel target for ELISA detection markers.
7. The application of the molecule combined with the serum polypeptide molecular diagnostic marker Ezrin in the preparation of the serum diagnostic reagent of brain glioma is characterized in that the amino acid sequence of the serum polypeptide molecular diagnostic marker Ezrin is shown in SEQ ID No. 1.
8. A diagnostic kit for brain glioma is characterized by comprising a serum polypeptide molecular diagnostic marker Ezrin, wherein the amino acid sequence of the Ezrin is shown in SEQ ID No. 1.
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