CN115097137A

CN115097137A - Screening method, application and kit of disease-related markers

Info

Publication number: CN115097137A
Application number: CN202210415007.4A
Authority: CN
Inventors: 邱晓彦; 段富刚; 朱珠; 戴慧; 张奕潇; 王一凡; 姜文华
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-11-11
Filing date: 2021-11-11
Publication date: 2022-09-23
Also published as: CN113777330A; CN113777330B

Abstract

The invention discloses a screening method, application and kit of disease-related markers. The screening method comprises the following steps: purifying immunoglobulin (Ig) complexes from the first healthy sample serum and the diseased sample serum, respectively, using a substance capable of binding Ig; performing protein mass spectrometry sequencing analysis on the protein combined with the Ig, comparing the difference between a healthy sample and a diseased sample, and finding out the difference protein only appearing in the diseased sample, namely the difference protein is the disease-related potential marker; finally, the potential marker was further verified: sera from the second healthy and diseased samples (expanded cases) were used, purified to obtain Ig complexes, and further characterized by antibodies specific for the different proteins. According to the method, an Ig compound (instead of whole serum) is obtained from serum, and then protein mass spectrum sequencing combined specific antibody analysis is carried out on the protein combined with the Ig, so that disease-related markers can be screened out quickly and effectively.

Description

Screening method, application and kit of disease-related markers

The application is a divisional application with the title of 'screening method, application and kit for disease-related markers' on application date 2021, 11/11, application number 202111329614.0.

Technical Field

The invention relates to the technical field of molecular biology and immunology, in particular to a screening method, application and a kit of disease-related markers.

Background

Biomarker (Biomarker) refers to an in vivo substance with objectively detectable and evaluable properties that can be used as an indicator of normal biological processes, pathological processes, or therapeutic intervention pharmacological responses. In the research of disease diagnosis and treatment, finding and discovering valuable biomarkers has become a research hotspot in the current medical field.

The patent publication No. CN108956791A discloses a method for large-scale screening of protein biomarkers: homogenizing and cracking a biological tissue control sample and a reference sample (pathological sample); carrying out operations such as enzymolysis and desalting on the lysate; performing protein identification by using a LC-MS/MS liquid chromatography-mass spectrometry (LC-MS/MS); analyzing the abundance change of the protein of the sample by comparing the mass spectrum analysis times or mass spectrum peak intensity, calculating the integral of the signal intensity of each peptide segment on LC-MS based on MS1, and correcting the data in large scale based on the identification result of MS 2; and screening protein biomarkers which are obviously up-regulated or down-regulated according to the corrected data analysis result.

The patent publication No. CN111999403A discloses a method for screening a serum marker of gas explosion, which comprises the steps of carrying out LC-MS detection on serum samples of noninvasive rats and gas explosion lung injury rats, analyzing by adopting an LC-MS serum metabonomics technology, constructing a model, carrying out data processing by using the model, and screening the biomarker of a lung injury model from candidate metabolites.

At present, the commonly used research for screening disease-related specific markers generally adopts complete genome data or gene expression data, complete serum protein data and the like as analysis bases. Taking the new coronary pneumonia as an example, the characteristic changes of proteins and metabolites in the serum of a severe patient COVID-19 are revealed through a protein mass spectrometry technology, and some marker molecules which may have application potential are identified. However, almost all studies looking for specific biomarkers utilize the whole serum protein of COVID-19. For protein mass spectrometry, because of the small sample size and the low serum or plasma content of the specific marker, it is obvious that the protein mass spectrometry, which requires a small sample size and is "dominant", can be called "sea fishing needle", i.e., it is not easy to select the meaningful marker from the target analytes.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a screening method, application and kit of potential biomarkers related to diseases, wherein the method can quickly screen out meaningful markers and provide a target basis for diagnosis and treatment of diseases.

In a first aspect, the present invention provides a method for screening potential markers associated with a disease, comprising:

(1) purifying the Ig complex from the first healthy sample serum and the diseased sample serum respectively by using a substance capable of binding Ig (immunoglobulin);

(2) performing mass spectrum analysis and/or sugar spectrum analysis on the Ig complex, comparing the analysis results of the healthy sample and the diseased sample, and finding out a differential protein which only appears in the Ig complex of the diseased sample in high frequency and is combined with the Ig, and/or a differential glycosyl which only appears in the Ig complex of the diseased sample in high frequency, wherein the differential protein and/or the differential glycosyl is a potential marker related to the disease;

(3) further expanding the number of healthy samples and diseased samples, purifying the obtained Ig complex by the same method as the step (1), and verifying the differential protein and/or the differential glycosyl.

Optionally or preferably, in the above method, the method for verifying is as follows:

identifying the differential protein in the Ig compound obtained in the step by using a specific antibody of the differential protein aiming at the differential protein, and further verifying that the differential protein is a potential marker related to the disease if the differential protein still appears or is deleted in high frequency in the diseased sample;

and (3) identifying the differential glycosyl of the Ig complex obtained in the step by using the conjugate of the glycosyl or sugar spectrum analysis aiming at the differential glycosyl, and further verifying that the differential glycosyl is a potential marker related to the disease if the differential glycosyl still appears or is deleted at high frequency in a diseased sample.

In the screening method, the healthy sample is a sample that can be distinguished from a diseased sample, for example, if it is desired to screen a marker related to a certain disease a, a sample of a normal patient who does not suffer from the disease a may be used as the healthy sample, or a sample of a person who suffers from another disease, for example, a disease B but does not suffer from the disease a may be used as the healthy sample, as long as it can be clearly distinguished from the disease and is not diagnosed as the disease.

The new batch of healthy samples and diseased samples are used in the verification step, the number of the new batch of healthy samples and diseased samples can be further enlarged and is larger than that of the first batch of healthy samples and diseased samples, due to the fact that the cost of mass spectrum or sugar spectrum analysis is high, the number of the first batch of samples only needs to be enough to find differential protein and/or differential glycosyl, after the differential protein and glycosyl are found, the marker characteristics of the differential protein and glycosyl are further verified in a large batch, the accuracy of the obtained result is better, and meanwhile the cost can be reduced.

In the above method, the high frequency refers to that the appearance or deletion frequency has a significant difference compared with other samples, i.e. the abundance shows a significant difference.

Ig, immunoglobulin, i.e. immunoglobulin. The substance capable of binding the Ig can be separated and purified after being combined with the Ig in serum, the compound contains other protein substances capable of binding with the Ig in the serum besides the Ig, and glycosylation modification possibly exists in partial proteins, so that the proteins capable of binding with the Ig are collected by using the substance capable of binding the Ig, and then the proteins are subjected to protein mass spectrum sequencing analysis and sugar spectrum analysis, and the difference between a healthy sample and a diseased sample is searched in the analysis result, so that the proteins and the sugar groups only appearing in the diseased sample at high frequency can be obtained. Finally, the feasibility of the differential protein serving as a disease-related marker is verified by an immunological binding technology such as ELISA, Western blot or CBA flow-type detection method, and the differential glycosyl can be further verified in a targeted manner.

Alternatively or preferably, in the above method, the substance capable of binding to Ig comprises at least one of protein g, protein a, Jacalin, Hitrap, anti-human IgD, anti-human IgE.

Alternatively or preferably, in the above method, the Ig complex comprises at least one of an IgG complex, an IgA complex, an IgM complex, an IgD complex, and an IgE complex.

In a second aspect, the present invention provides the use of a differential protein and/or a differential sugar moiety as potential markers in the manufacture of a medicament for the diagnosis or treatment of a disease, said differential protein and/or differential sugar moiety being screened by any of the methods described above.

Alternatively or preferably, in the above application, the disease includes any one of infectious diseases and immune-related diseases.

In a third aspect, the invention provides a detection kit for assisting in detecting neocoronary pneumonia, which is used in combination with a substance capable of binding to Ig, wherein the kit comprises at least one of an anti-CA 1 antibody, an anti-LRG 1 antibody, and an anti-IgG-IgA complex antibody.

In a fourth aspect, the invention provides a detection kit for assisting in detecting brain glioma, wherein the kit is used in combination with a substance capable of binding to Ig, and is characterized in that the kit comprises an anti-S100-A8 antibody. S100-A8 is Protein S100-A8 hereinafter.

Compared with the prior art, the invention has the following beneficial effects:

the screening method comprises the steps of purifying an Ig compound from sample serum by utilizing a substance capable of combining Ig based on an affinity purification technology, taking a protein combined with the Ig in the Ig compound as a research object of a protein mass spectrum sequencing technology, analyzing the difference of different proteins in a healthy sample and a diseased sample by sequencing, further searching for the protein only appearing in the serum of the diseased sample, or carrying out sugar spectrum analysis on the Ig compound to find out the difference glycosyl, even if the concentration of the components in the serum is very low, the screening method can also quickly and effectively screen the components, and the components can be verified to be disease-related markers after the identification of the proteins by an immunological technology and the identification of the glycosyl.

Whether exogenous antigens (such as various viruses, bacteria and fungi) or intracellular antigen release caused by various diseases, trauma and the like, can activate the immune system in vivo, which can achieve immune defense by specifically recognizing and binding antigenic substances to eliminate antigens. For example, antigenic substances can activate B cells to produce specific antibodies that in turn exert an immune clearance effect by binding to a specific antigen. Therefore, under the disease state, the antigen combined by some antibodies can have the molecular characteristics related to the disease, the molecules can be the components of pathogenic microorganisms directly causing the disease, and can also be the components of endogenous antigens after tissue damage. In addition, the sugar spectrum analysis result of the Ig complex shows that the healthy sample and the diseased sample have significant difference, so that the related diseases can be distinguished by using the sugar radicals with the difference. The mode of firstly forming the Ig complex and then searching for the difference based on the Ig complex obviously reduces the screening range, saves the time and the capital cost and has higher operability.

The new coronary pneumonia related markers CA1, LRG1 and IgG-IgA compound screened by the method can specifically identify the new coronary pneumonia, can be used as a biomarker of the new coronary pneumonia, has important significance for revealing pathogenesis of diseases such as immune damage mechanism of the new coronary pneumonia, and can provide effective targets for related disease treatment.

Drawings

FIG. 1 is a schematic diagram of the procedure and principle of example 1-identification of Ig-binding proteins in serum. A is a flow chart and B is a schematic diagram. And affinity purifying the Ig compound combined with IgA, IgG and IgM by using Jacalin, Protein G or anti-human IgM affinity chromatographic columns respectively, and performing Ig binding Protein mass spectrum identification and data analysis. HC: healthy group. CP (2 weeks): the convalescent period of the new coronary pneumonia is 2 weeks.

FIG. 2 is a graph showing the results of Western blot method of example 1 to identify that CA1 and LRG1 are specifically present in Ig complex of patients with new coronary pneumonia. A is the level of IgA binding to CA1 in the sera of the different groups. B is the level of IgG binding to LRG1 in different groups of sera. Healthy control indicates Healthy, i.e., normal, group, COVID-19 conditional Phase (2weeks) indicates the COVID-19 recovery period of 2weeks, COVID-19 vaccination indicates the COVID-19 vaccination group, and COVID-19 conditional Phase (6months) indicates the COVID-19 recovery period of 6 months.

FIG. 3 is a graph showing the identification of IgG-IgA complexes specifically present in Ig complexes of patients with new coronary pneumonia according to the Western blot method of example 1. A: the level of IgA to IgG binding in serum of healthy groups, COVID-19convalescent period of 2weeks, COVID-19convalescent period of 6months, and COVID-19 vaccinated groups was identified. B: the level of IgG to IgA binding in serum of healthy groups, COVID-19convalescent period of 2weeks, COVID-19convalescent period of 6months, and COVID-19 vaccinated groups was identified.

FIG. 4 is a graph showing the difference in mass spectrometry between IgG-binding proteins of patients with coronary heart disease and normal persons in example 2, wherein CH represents patients with coronary heart disease and NM represents normal persons.

FIG. 5 is the difference of mass spectrometry of IgG-binding protein between acute myocardial infarction and normal human in example 2, wherein AM represents acute myocardial infarction and NM represents normal human.

FIG. 6 is the difference of mass spectrometry of IgG-binding protein between acute myocardial infarction and coronary heart disease in example 2, wherein AM represents acute myocardial infarction patient and CH represents coronary heart disease patient.

FIG. 7 is a graph showing the difference in mass spectrometry between IgG-binding proteins of three groups of patients with acute myocardial infarction, coronary heart disease and normal persons in example 2, AMI for acute myocardial infarction, CHD for coronary heart disease and NM for normal persons.

FIG. 8 is a graph showing the comparison of the differences in expression of Protein S100-A8 after mass spectrometry of IgG-binding proteins of four groups of patients with lung cancer, brain glioma, gastrointestinal inflammation and coronary heart disease in example 3.

FIG. 9 is a graph comparing the differences in Fibronectin expression after mass spectrometry of IgG-binding proteins in four groups of the population of example 3.

FIG. 10 is a graph comparing the differences in expression of Suprabasen after mass spectrometric analysis of IgG binding proteins in the four populations of example 3.

FIG. 11 is a graph comparing the differences in expression of Annexin A2 after mass spectrometry of IgG binding proteins in four populations of example 3.

Fig. 12 is a graph of the mass spectra of IgG-binding proteins in four groups of the population in example 3, analyzed by T-test after being divided into cancer and cancer groups, showing that 5 proteins have significantly different abundances in the cancer and non-cancer groups.

FIG. 13 is a graph comparing the differences in expression of sialic acid (glycosylation) modified glycoproteins shown after glycoprofiling of IgG binding proteins in four groups of humans in example 3.

FIG. 14 is a graph comparing the differences in expression of G2 FS-modified glycoproteins after completion of glycoprofiling of IgG-binding proteins in four groups of population in example 3.

FIG. 15 is a graph comparing the differences in expression of G1S-modified glycoproteins after completion of glycoprofiling of IgG-binding proteins in four groups of population in example 3.

FIG. 16 is a graph comparing the differences in expression of G1 FS-modified glycoproteins after completion of glycoprofiling of IgG-binding proteins in four groups of population in example 3.

FIG. 17 is a graph comparing the differences in expression of G2S-modified glycoproteins after completion of glycoprofiling of IgG-binding proteins in four groups of population in example 3.

FIG. 18 is the results of the immunobinding assay of Protein S100-A8 as a potential brain glioma-associated marker in example 3, showing a higher frequency of expression in the brain glioma group.

Detailed Description

The technical solution of the present invention will be explained and illustrated in detail with reference to preferred embodiments.

Example 1 screening of potential markers associated with neocoronary pneumonia

First, purification of immunoglobulin complexes and protein mass spectrometry.

Respectively taking 200 mu L of 9 new coronary pneumonia patients (recovery period 2weeks) and 9 healthy human serums, diluting the serum by 10 times by PBS, then respectively using Jacalin, Protein G and anti-human IgM, carrying out affinity purification on IgA complexes, IgG complexes and IgM complexes in the serum by an affinity chromatography column, and then using the three Ig complexes for Protein mass spectrum sequencing analysis, wherein the operation flow is shown in figure 1.

Jacalin is capable of affinity purifying IgA in serum, and further purifying various proteins bound with IgA. Protein G can perform affinity purification on IgG in serum, and further purify various proteins bound to the IgG together. The anti-human IgM can perform affinity purification on IgM in the serum, and further purify various proteins bound with IgM together.

The following results were obtained by further analysis of the protein mass spectrum data:

TABLE 1 IgA Complex IgA binding proteins

Protein present only in patients with new coronary pneumonia (2weeks of convalescence).

Referring to table 1, 42 proteins bound to IgA increased and 1 protein decreased in the neocoronary pneumonia group compared to the healthy group, with CA1 and SERPINA6 appearing only in 9 patients with neocoronary pneumonia.

Further enrichment analysis of the differential proteins revealed that proteins that bind IgA are highly abundant in the processes of inflammation, complement system and coagulation cascade.

Proteins binding to IgG in IgG complexes

As can be seen from table 2, the new coronary pneumonia group has 22 protein increases and 6 protein decreases in IgG-binding protein compared to the healthy group, wherein LRG1 appears in 8 new coronary patients only, and the protein binding to IgG is enriched in the negative regulatory pathways of complement activation, endocytosis and proteolysis.

Proteins binding to IgM in the IgM complexes

Referring to table 3, the new coronary pneumonia group showed an increase in 6 proteins bound to IgM and a decrease in 7 bound proteins compared to the healthy group, but no proteins were found which were present only in patients with new coronary pneumonia.

In the second part, the Western blot method was used to further identify that CA1 and LRG1 were specifically present in the immunoglobulin complex of neocorona patients.

Candidate markers selected in the first part were identified for CA1 and LRG 1. CA1, Carbonic Anhydrase I, Carbonic Anhydrase I. LRG1, leucine-rich α 2-glycoprotein 1, leucine-rich-alpha-2-glycoproten 1.

We further expanded the sample size to detect 20 healthy persons, 25 COVID-19 convalescence stages for 2weeks, 20 COVID-19 convalescence stages for 6months and 4 COVID-19 vaccinees sera, affinity-purified the IgA bound immunoglobulin complexes using a Jacalin affinity chromatography column, and the resulting immunoglobulin complexes detected by immunoblotting using anti-CA 1 antibody. IgG complexes were affinity purified using Protein G affinity chromatography column and detected by immunoblotting with anti-LRG 1 antibody.

The detection result is shown in FIG. 2A, B. The results found that CA1 and LRG1 were, without exception, found only in the immunoglobulin complexes obtained from all patients sera at 2weeks of convalescence of covd-19, but not in other normal persons (healthy group), vaccinees and samples of covd-19 patients 6months after recovery, indicating that CA1 and LRG1 could be potential markers of new coronary pneumonia.

In addition, we have identified immune complexes that are specific for IgG-IgA complexes that contain both IgG and IgA immunoglobulins in neonatal patients.

Occasionally, the protein profile of the immunoglobulin complex bound to IgA was analyzed, which contained a certain amount of IgG, whereas the immunoglobulin complex bound to IgA in the serum of healthy persons was lacking IgG.

To demonstrate that IgA-bound immunoglobulin complexes do contain IgG (possibly as a new marker molecule) in serum from patients with new coronary pneumonia, we further expanded the samples and collected 15 healthy people, 20 patients with new coronary recovery period of 2weeks, 15 COVID-19 recovery period of 6months and 4 sera from COVID-19 vaccinees and Western blot analysis was performed on the Jacalin affinity purified IgA-bound immunoglobulin complexes with anti-human IgG.

As a result, IgG was found without exception only in all patients with a convalescent covd-19 period of 2weeks, but not in other normal groups, vaccinees and the covd-19 patients group 6months after recovery, see fig. 3A.

Similarly, we tested affinity purified immunoglobulin complexes that bind IgG for the presence of IgA using anti-IgA antibodies.

As a result, IgA was found without exception only in all COVID-19convalescent patient groups at 2weeks, but not in other normal groups, the vaccinator group and the COVID-19 patient group 6months after recovery, see FIG. 3B. The IgG-IgA complex is specifically present in the immunoglobulin complex of the new coronary patient and can be used as a potential marker of the new coronary pneumonia.

Example 2 screening of potential markers associated with Coronary Heart Disease (CHD) and Acute Myocardial Infarction (AMI)

A first part: screening for potential Coronary Heart Disease (CHD) associated markers

Purification of IgG complex and protein mass spectrometry: serum from patients with Coronary Heart Disease (CHD) and serum from normal human (NM) were separately diluted with PBS and subjected to affinity purification of IgG complexes in the serum using Protein G through an affinity column. Mass spectrometry analysis was performed on proteins bound to IgG.

The results of mass spectrometry of proteins are shown in FIG. 4, in which the names of the proteins represented by the different protein markers are shown in the following table:

numbering	Gene	English full scale	Chinese full scale
				P02679	FGG	Fibrinogen gamma chain	Fibrinogen gamma chain
P02671	FGA	Fibrinogen alpha chain	Fibrinogen alpha chain
				P08493	MGP	Matrix Gla protein	Extracellular matrix Gla proteins
P01023	A2M	Alpha-2-macroglobulin	Alpha-2 macroglobulin
				P02774	GC	Vitamin D-binding protein	Vitamin D binding proteins
P03951	F11	Coagulation factor XI	Blood coagulation factor 11
				P02655	APOC2	Apolipoprotein C-II	Apolipoprotein C-II
P02765	AHSG	Alpha-2-HS-glycoprotein	alpha-2-HS glycoproteins
				P02656	APOC3	Apolipoprotein C-III	Apolipoprotein C-III
Q96HU8	DIRAS2	GTP-binding protein Di-Ras2	GTP-binding protein Di-Ras2
				P31947	SFN	14-3-3protein sigma	14-3-3sigma

The results showed that fibrinogen, extracellular matrix Gla protein, alpha-2 macroglobulin, vitamin D binding protein, coagulation factor 11, apolipoprotein C-II, alpha-2-HS glycoprotein, apolipoprotein C-III, GTP binding protein Di-Ras2 and 14-3-3sigma are present in CHD patient sera at high levels compared to normal humans. The protein can be used as a potential marker related to CHD.

A second part: screening of potential markers associated with Acute Myocardial Infarction (AMI)

Purification of Ig complex and protein mass spectrometry: serum of Acute Myocardial Infarction (AMI) patients and serum of normal human (NM) are respectively taken, diluted by PBS, and then purified by ProteinG from the serum of the Acute Myocardial Infarction (AMI) and the normal human (NM) to obtain IgG immune complex, and the protein combined with the IgG is subjected to mass spectrometry to search for differences.

Results of the analysis see FIG. 5, in which the names of the proteins represented by the different protein markers are shown in the following table:

the results showed that fibrinogen, IGHV4-30-2, IGHV4-4, apolipoprotein C-III, ATP-dependent RNA helicase A, coagulation factor 10, IGKV1-6 and IGKV3-20 were present at high levels in the sera of AMI patients compared to normal humans. Indicating that the protein can be used as a potential marker related to AMI.

Coronary Heart Disease (CHD) is caused by myocardial ischemia due to atherosclerotic lesions of coronary arteries, and if coronary heart disease is not treated effectively and allowed to develop, acute necrosis of part of the myocardium may occur, which translates into Acute Myocardial Infarction (AMI). Therefore, to further look for the difference between CHD and AMI and accurately determine the progression of the disease process, we compared the protein binding to IgG in CHD and AMI patients by mass spectrometry.

And a third part: further screening for potential markers associated with CHD and AMI

Purification of Ig Complex and protein Mass Spectrometry: serum of patients with Coronary Heart Disease (CHD) and polar myocardial infarction (AMI) is respectively taken, diluted by PBS, and affinity purification is carried out by Protein G through an affinity chromatography column to obtain IgG compound in the serum. The proteins bound to IgG in the complexes were subjected to mass spectrometry.

The results of mass spectrometry of proteins are shown in FIG. 6, in which the names of the proteins represented by the different protein markers are shown in the following table:

number of	Gene	English full scale	Chinese full scale
				P09871	C1S	Complement C1s subcomponent	Complement C1S
P01602	IGKV1-5	Immunoglobulin kappa variable 1-5	IGKV1-5
				P00736	C1R	Complement C1r subcomponent	Complement C1R
P00742	F10	Coagulation factor X	Blood coagulation factor 10
				A0A0C4DH73	IGKV1-12	Immunoglobulin kappa variable 1-12	IGKV1-12
A0A0C4DH67	IGKV1-8	Immunoglobulin kappa variable 1-8	IGKV1-8
				P01742	IGHV1-69	Immunoglobulin heavy variable 1-69	IGKV1-69
A0A0C4DH55	IGKV3D-7	Immunoglobulin kappa variable 3D-7	IGKV3D-7
				A0A0C4DH25	IGKV3D-20	Immunoglobulin kappa variable 3D-20	IGKV3D-20
P0DOX5		Immunoglobulin gamma-1heavy chain	Immunoglobulin gamma-1 heavy chain
				A0A087WSY6	IGKV3D-15	Immunoglobulin kappa variable 3D-15	IGKV3D-15

The results show that AMI patients present high levels of complement C1S, C1R, coagulation factor 10, and IGKV1-5 compared to CHD. And as a result of combining the first and second portions, AMI and CHD both highlighted fibrinogen and apolipoprotein C-III compared to the normal human IgG complex protein mass spectrum. The fibrinogen and the apolipoprotein C-III can be used as potential markers of CHD and AMI, and the specific disease of the patient and the progress of the disease can be accurately judged by combining the potential markers of CHD and AMI screened by the first part and the second part.

Fig. 7 shows the results of mass spectrometry analysis of proteins bound to IgG in three groups of Acute Myocardial Infarction (AMI), Coronary Heart Disease (CHD) and Normal (NM), showing differences in protein profiles of the three groups.

Example 3 screening of brain glioma-associated markers

Purification of Ig complex and protein mass spectrometry: four groups of serum of patients with brain glioma, patients with coronary heart disease, patients with lung cancer and patients with gastrointestinal cancer disease, wherein 10 persons in each group are respectively diluted by PBS and then affinity purified by Protein G through an affinity chromatography column. The proteins in the resulting IgG complexes were subjected to mass spectrometry and glycospectrometry.

Referring to fig. 8 and 9, the results of mass spectrometry showed that both Protein S100-A8(5/10) and fibrinectin (3/10) were expressed at high levels in IgG complexes purified from blood samples of brain glioma patients compared to the other groups. Referring to fig. 10 and 11, the sera of lung cancer patients showed particularly high frequency of suprabassin (5/10, i.e. 5 out of 10 samples showed high expression) and Annexin a2 (Annexin a2) (4/10).

Analysis by the T-test revealed that, referring to FIG. 12, when the "T-test Difference" was greater than 1.5 or less than-1.5, the abundance of the protein was significantly different between the two groups. The software analyzed five different proteins: c4b-binding Protein alpha chain (C4b binding Protein. alpha. -chain), 2.Ig alpha-2chain C region (Ig. alpha. -2chain C region), 3.Ig kappa chain V-II FR (Ig. kappa. chain V-II FR), 4.Ig mu heavy chain disease Protein (Ig-. mu.H chain disease Protein), 5.Ig lambda chain V region 4A (Ig. lambda. chain V region 4A).

Among them, Protein profile showed that Ig lambda chain V region 4A was at a much higher level in the cancer group than in the non-cancer group (coronary heart disease) while C4b-binding Protein alpha chain Ig alpha-2chain C region, Ig kappa chain V-II FR and Ig mu fatty chain disease Protein were significantly higher in the non-cancer group than in the cancer group. This indicates that the five proteins can be used as potential cancer-related markers, and the significant increase or decrease of the expression level of the five proteins relative to the non-cancer control sample indicates the possibility of cancer.

Referring to fig. 13, the results of glycoprofiling showed that sialic acid (sialylation) also exhibited high levels in brain glioma patients. In addition, referring to fig. 14 and 15, G2FS and G1S also exhibited high levels in patients with brain glioma. With reference to fig. 16, G1FS exhibited high levels in lung cancer patients, and with reference to fig. 17, G2S exhibited high levels in lung cancer patients. In each figure, the squares in the glycosyl structure represent N-acetylglucosamine (N-acetylglucosamine), the light gray circles represent galctose (galactose), the dark gray circles represent mannose (mannose), the diamonds represent sialic acid (sialic acid), and the triangles represent fucose (fucose).

Through the mass spectrum analysis and the sugar spectrum analysis, Protein S100-A8, fibrinectin, sialic acid, G2FS and G1S can be used as potential markers related to brain glioma, and G1FS and G2S can be used as potential markers related to lung cancer.

When sialic acid is verified, it can be detected by Sambucus nigra lectin (SNA) or Mal, and other sugar groups can be detected by sugar profiling.

Further validation experiments were also performed for Protein S100-A8.

Expanding the number of samples, selecting 28 new brain glioma patient samples and 27 new coronary disease patient samples, respectively collecting serum, and performing affinity purification by using Protein G through an affinity chromatography column to obtain IgG compounds in the serum.

The IgG compound obtained by the method is subjected to an immune binding experiment by using a specific antibody against Protein S100-A8, and the result is shown in figure 18, wherein the Protein S100-A8 has higher-frequency expression in a brain glioma group, and has obvious difference with a coronary heart disease group, so that the Protein S100-A8 can be further verified to be used as a brain glioma related marker for assisting in diagnosing the brain glioma.

The inventive concept is explained in detail herein using specific examples, the above description of which is only intended to facilitate the understanding of the core concepts of the present invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are intended to be included within the scope of the present invention.

Claims

1. A method of screening for potential markers associated with a disease, comprising:

(1) purifying the Ig complex from the first healthy sample serum and the diseased sample serum respectively by using a substance capable of binding Ig;

(2) performing mass spectrum analysis and/or sugar spectrum analysis on the Ig complex, comparing the analysis results of the healthy sample and the diseased sample, and finding out a differential protein which only appears or lacks in the Ig complex of the diseased sample at a high frequency and is combined with the Ig, and/or a differential glycosyl which only appears or lacks in the Ig complex of the diseased sample at a high frequency, wherein the differential protein and/or the differential glycosyl is a potential marker related to the disease;

2. The method of claim 1, wherein the authentication method is:

aiming at the differential protein, utilizing a specific antibody of the differential protein to identify the differential protein in the Ig compound obtained in the step, and if the differential protein in the diseased sample still appears or is deleted in high frequency, further verifying that the differential protein is a potential marker related to diseases;

3. The screening method according to claim 1, wherein the substance capable of binding Ig comprises at least one of ProteinG, ProteinA, Jacalin, Hitrap, anti-human IgD, anti-human IgE.

4. The screening method of claim 1, wherein the Ig complex comprises at least one of an IgG complex, an IgA complex, an IgM complex, an IgD complex, and an IgE complex.

5. Use of a differential protein and/or a differential sugar moiety as a potential marker in the manufacture of a medicament for the diagnosis or treatment of a disease, wherein the differential protein and/or the differential sugar moiety is selected by the method of any one of claims 1 to 4.

6. The use according to claim 5, wherein the disease comprises any one of infectious diseases and immune-related diseases.

7. The use according to claim 6, wherein the disease comprises any one of neocoronary pneumonia, lung cancer, brain glioma;

when the disease is neocoronary pneumonia, the differential protein comprises at least one of CA1, LRG1 and IgG-IgA complex;

when the disease is lung cancer, the differential protein comprises at least one of supra, annexin A2, and the differential glycosyl comprises at least one of G2S and G1 FS;

when the disease is brain glioma, the differential Protein comprises at least one of Protein S100-A8 and fibronectin, and the differential glycosyl comprises at least one of sialic acid, G2FS and G1S.

8. A detection kit for assisting in detecting neocoronary pneumonia, the kit being used in combination with a substance capable of binding Ig, wherein the kit comprises at least one of an anti-CA 1 antibody, an anti-LRG 1 antibody, and an anti-IgG-IgA complex antibody.

9. The detection kit according to claim 8, further comprising a mass spectrometry result reference of an Ig complex of a new coronary pneumonia patient and/or a mass spectrometry result reference of an Ig complex of a healthy human.

10. A detection kit for the auxiliary detection of brain glioma, used in combination with a substance capable of binding Ig, characterized in that the kit comprises an anti-S100-A8 antibody.