CN114354930A - Application of serum polypeptide diagnostic marker AFP of brain glioma - Google Patents

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 molecule diagnostic marker AFP is shown in SEQ.ID.NO.1, the AFP is human alpha-fetoprotein, and the accurate molecular weight of the AFP is 3318.82 daltons. The AFP presents specific high expression in the serum detection of brain glioma patients, and the expression level of the AFP is detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ELISA method, so that the AFP can be used as the detection method of the serum of brain glioma patients.

Description

Application of serum polypeptide diagnostic marker AFP of brain glioma

Technical Field

The invention belongs to the technical field of biomarkers, and particularly relates to a serum polypeptide diagnostic marker AFP 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. So far, no effective method for curing the disease exists, and surgical excision and early chemoradiotherapy can prolong the life of a patient.

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 tumor markers in blood, particularly protein markers in the blood, thereby realizing 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 Alpha-fetoprotein (AFP) which 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 AFP in the preparation of a brain glioma serum diagnostic reagent, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker AFP is shown in SEQ.ID.NO. 1.

Preferably, the molecular weight of the serum polypeptide molecular diagnostic marker AFP is 3318.82 daltons.

Preferably, the detection parameter of the serum polypeptide molecular diagnostic marker AFP in the serum of a patient with brain glioma is 56.785-166.385pg/mL, and the detection parameter in the serum of a normal healthy population is 43.81-116.85 pg/mL.

Preferably, the serum polypeptide molecular diagnostic marker AFP is significantly highly expressed in serum samples of patients with brain glioma compared to serum samples of normal healthy people.

Preferably, the brain glioma serum diagnostic marker is a serum polypeptide molecule for detecting brain glioma by ELISA.

Preferably, the serum polypeptide molecule diagnostic marker AFP is a novel target for ELISA detection of markers.

The invention also discloses application of a molecule combined with the serum polypeptide molecular diagnostic marker AFP in preparing a serum diagnostic reagent for brain glioma, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker AFP 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 AFP, wherein the amino acid sequence of the AFP 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 AFP. AFP is human alpha-fetoprotein, which has an exact molecular weight of 3318.82 daltons. AFP shows specific high expression in serum detection of brain glioma patients: the expression range in the serum of normal healthy people is as follows: 43.81-116.85 pg/mL; the expression range in the serum of patients with brain glioma is as follows: 56.785-166.385pg/mL, and there was a very significant difference in expression between the two groups (p < 0.01).

The AFP can be used as a diagnostic marker of the brain glioma serum in view of the specific high expression of the AFP in the brain glioma serum; the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) is used for detecting AFP or the ELISA method is used for detecting the expression level of AFP, and the method can be used as a method for detecting patients with brain glioma. And aiming at ELISA detection of brain glioma serum diagnosis, AFP can be used as a new target point of an ELISA detection reagent.

Drawings

FIG. 1 is a graph of the difference in protein polypeptide expression of the protein polypeptide peak m/z:3318.82 in brain glioma patients (red) and in a normal healthy population group (green) according to the present invention;

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 of AFP of the present invention; wherein, m/z:3318.82, z is 3;

FIG. 4 is a graph showing the expression level of AFP protein in serum of patients and normal healthy persons of glioma of the present invention; wherein, the glioma group N is 34, and the normal healthy population N is 28.

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 diagnosis marker, has specificity in expression, and can be applied to brain glioma diagnosis. The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The screening method of the serum diagnosis marker of the brain glioma 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 the difference of the serum protein polypeptide spectrograms of the serum protein polypeptides of the brain glioma patient and 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 from a protein polypeptide peak with significant low expression in the serum of the brain glioma patient.

The method for verifying the screened brain glioma polypeptide serum diagnostic marker comprises the following steps:

the protein polypeptide mixture separated from the serum of a patient with brain glioma is divided into 20-30 components by using HPLC (high performance liquid chromatography), secondary mass spectrum identification is carried out on the protein polypeptide mixture, an enzyme-linked immunosorbent assay is carried out on the identified protein polypeptide, and serum regression verification results prove that the protein polypeptide mixture is remarkably high-expressed in the serum of the patient with brain glioma, has specificity and can be used as a biomarker for screening the serum of the patient with brain 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 L of MB-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;

fifthly, adding 5 mu L of MB-WCX eluent to wash the 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 centrifugal tube;

sixthly, adding 5 mu L of MB-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 24 hours for mass spectrometry.

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
				3318.82↑	<0.000001	6.64±3.07	3.85±1.37

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:3318.82 in brain glioma patients (red) compared to normal healthy population (green), m/z:3318.82 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: 3318.82, and the mass spectrum collected by magnetic bead separation is loaded by adopting a nano liter flow rate HPLC liquid phase system Easy nLC 1200 of Thermo Scientific company, the rest serum protein polypeptide is subjected to two-dimensional gel chromatography, and then the expression of the protein polypeptide m/z up-regulated in the serum of a patient with brain glioma is carried out by combining with an Orbitrap Fusion mass spectrometer of Thermo Scientific: 3318.82 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 C18trap column (C183 m 0.10X 20mm) by an autosampler 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

Time (min)	A(0.1％FA/H2O)	B(0.1％FA/ACN)	Flow rate (nL/min)
				0	92％	8％	600
8	88％	12％	600
				48	77％	23％	600
68	64％	36％	600
				69	5％	95％	600
78	5％	95％	600

3.3 Mass Spectrometry identification

Each sample was separated by capillary hplc and analyzed by mass spectrometry using Orbitrap Fusion mass spectrometer (Thermo scientific) with the main parameter settings shown in table 3 and the mass spectrometric profile shown in fig. 3.

Table 3 main parameter settings for mass spectrometry

3.4 data analysis

Analyzing the RAW data into RAW files by mass spectrometry, performing library searching identification in a database of unidrop-human by using software sequence and a protocol discover 2.5.0(Thermo Scientific), submitting the RAW files to a sequence server through the protocol discover during library searching, selecting the well-established database, and then searching the database, 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, the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) is used for detecting AFP or the ELISA method is used for detecting the expression level of AFP, and the method can be used as a method for detecting patients with brain glioma. AFP was suggested to be a protein specifically associated with brain glioma and was further validated by ELISA detection.

4. ELISA serum validation analysis of brain glioma serum AFP expression

4.1 serum samples

Serum verification analysis by ELISA was performed by collecting 34 cases of serum from patients with brain glioma (16 cases for male; 18 cases for female) and 28 cases of serum from normal healthy population (13 cases for male; 15 cases for female). 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 AFP 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 alpha-fetoprotein (AFP) in a specimen by using a double-antibody sandwich method. Coating a microporous plate with a purified human alpha-fetoprotein (AFP) antibody to prepare a solid-phase antibody, sequentially adding the alpha-fetoprotein (AFP) into the micropores coated with the monoclonal antibody, combining with the HRP-labeled alpha-fetoprotein (AFP) 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 is positively correlated with alpha-fetoprotein (AFP) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the concentration of human alpha-fetoprotein (AFP) 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 AFP in different detection groups is greater than that of normal healthy population of brain glioma patients, and the two groups have significant difference, and the specific results are shown in Table 6 and FIG. 4:

TABLE 6 expression levels of AFP protein in sera of different groups

ELISA detection is carried out on AFP in serum of patients with brain glioma and normal healthy people, and the result shows that the expression of AFP has specificity: the range of expression in serum in brain glioma patients was: 56.785-166.385 pg/mL; the range of expression in serum in normal healthy people is: 43.81-116.85pg/mL, and there was a very significant difference in expression between the two groups (p < 0.01). This indicates that: AFP is closely related protein of a patient with brain glioma, and can be used as a primary detection index of brain glioma lesion.

Therefore, the AFP expression of the serum sample to be tested can be preliminarily judged to be a brain glioma patient (56.785-166.385pg/mL) or a normal healthy human (43.81-116.85pg/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

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<141> 2021-11-25

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Claims (8)

1. The application of the serum polypeptide molecular diagnostic marker AFP 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 AFP is shown in SEQ.ID.NO. 1.

2. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker AFP has a molecular weight of 3318.82 daltons.

3. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker AFP is detected in serum of patients with brain glioma at a parameter of 56.785-166.385pg/mL and in serum of normal healthy people at a parameter of 43.81-116.85 pg/mL.

4. The use according to claim 1, wherein the serum polypeptide molecule diagnostic marker AFP is significantly 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 molecule diagnostic marker AFP is a novel target for ELISA detection markers.

7. The application of the molecule combined with the serum polypeptide molecular diagnostic marker AFP 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 AFP is shown in SEQ.ID.NO. 1.

8. A diagnostic kit for brain glioma is characterized by comprising a serum polypeptide molecular diagnostic marker AFP, wherein the amino acid sequence of the AFP is shown in SEQ.ID.NO. 1.

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