CN107765017B

CN107765017B - Markers for breast cancer

Info

Publication number: CN107765017B
Application number: CN201710931676.6A
Authority: CN
Inventors: 梁一; 关鑫; 黄壮霖
Original assignee: Guangdong Medical University
Current assignee: Guangdong Medical University
Priority date: 2017-10-09
Filing date: 2017-10-09
Publication date: 2020-01-10
Anticipated expiration: 2037-10-09
Also published as: CN107765017A

Abstract

The invention relates to the field of biomedical detection, and provides a breast cancer marker. The markers include at least one of the following glycoproteins: serum transferrin having a terminal N-acetylglucosamine glycosyl, a plasma protease C1 inhibitory factor having a terminal N-acetylglucosamine glycosyl, and the serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl. The amount of any one, two or all three of the three proteins in the sample to be detected is detected, so that the occurrence and/or the development of the breast cancer can be judged or assisted to be judged. The several/similar proteins can be used as a marker for breast cancer screening or auxiliary screening diagnosis singly or in any combination.

Description

Markers for breast cancer

Technical Field

The present invention relates to the field of biomedicine, in particular, to a disease detection marker, and more particularly, to a breast cancer marker.

Background

Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast gland. The incidence of breast cancer worldwide has been on the rise since the end of the 70 s of the 20 th century. According to data of breast cancer onset published by the national cancer center and the health department disease prevention and control agency in 2012, the data show that: the incidence of breast cancer in tumor registration areas nationwide is on the 1st of female malignant tumors.

According to the seventh edition of the united states committee for cancer committee for clinical staging of breast cancer, AJCC, breast cancer can be classified as Stage 0: TisN0M 0; stage I: T1N0M 0; stage II A: T0N1M0, T1N1M0, T2N0M 0; stage II B: T2N1M0, T3N0M 0; StageIIIA: T0N2M0, T1N2M0, T2N2M0, T3N1-2M 0; StageIIIB: T4N0-2M 0; StageIIIC: any TN3M 0; StageIV: any T any N Mlstage. Where Tis refers to carcinoma in situ, T represents the primary tumor, N represents regional lymph nodes, and M represents distant metastasis.

Breast cancer in situ is not fatal; however, the breast cancer cells lose the characteristics of normal cells, and the cells are loosely connected and easily fall off. Once cancer cells are shed, free cancer cells can be disseminated to the whole body along with blood or lymph fluid to form metastasis, which endangers life.

The breast cancer screening and monitoring, particularly the early screening, auxiliary diagnosis and diagnosis, is beneficial to the early discovery and tracking judgment of the breast cancer, can improve the success rate of curing the breast cancer, and has great significance.

Disclosure of Invention

The present invention aims to solve at least one of the above technical problems to at least some extent or to at least provide a useful commercial choice.

According to a first aspect of the present invention there is provided a marker for breast cancer, said marker comprising at least one of the following glycoproteins: serum transferrin (Serotransferrin) having a terminal N-acetylglucosamine glycosyl group, Plasma protease C1inhibitor (Plasma protease C1inhibitor) having a terminal N-acetylglucosamine glycosyl group, and serine protease inhibitor B4(Serpin B4) having a terminal N-acetylglucosamine glycosyl group.

The three glycoproteins with specific glycosyl structures are obtained by identifying glycoproteins differentially expressed in plasma of breast cancer patients by using Agrocybe cylindracea lectin 2 (AAL 2) which is found and separated earlier by the research team and a method combining lectin affinity chromatography with high performance liquid chromatography/mass spectrometry, and further screening and testing. The inventor finds that the three glycoproteins with specific glycosyl structures are differentially expressed in breast cancer patients and even breast cancer patients at different periods respectively, detects the concentration of any one, two or all three of the three proteins in a sample to be detected, can judge or assist in judging the occurrence and/or the development of the breast cancer, and can be used as a marker for screening or assisting in screening diagnosis of the breast cancer by the determined proteins alone or in any combination.

According to an embodiment of the invention, the marker is a glycoprotein. In one particular example, the marker is serum transferrin having a terminal N-acetylglucosamine glycosyl group. In another specific example, the marker is a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl. In yet another specific example, the marker is serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl group.

According to an embodiment of the invention, the marker is a composition, consisting of a plurality of glycoproteins. In one particular example, the markers are composed of serum transferrin having a terminal N-acetylglucosamine glycosyl group and a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl group. In one particular example, the markers referred to are protein compositions consisting of serum transferrin having a terminal N-acetylglucosamine glycosyl and the serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl. In one particular example, the markers are protein compositions consisting of a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl and a serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl. In one particular example, the markers referred to are protein compositions consisting of serum transferrin having a terminal N-acetylglucosamine glycosyl, a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl, and the serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl.

According to an embodiment of the present invention, the marker has a terminal N-acetylglucosamine glycosyl group which is a non-reduced terminal N-acetylglucosamine glycosyl group.

According to an embodiment of the invention, the said marker is capable of specifically binding to the lectin AAL 2. The sugar chip experiment of AAL2 shows that The highest sugar binding specificity is terminal N-acetylglucosamine (N-acetylglucosamine, GlcNAc) [ Jiang S, Chen Y, Wang M, et al.A novel selection from agarose gel maker show that The highest sugar binding specificity is terminal N-acetylglucosamine [ J ]. The biochemical journal, 2012, 443 (2): 369-378], AAL2 has higher specificity for GlcNAc than other lectins that bind GlcNAc, and is known to bind GlcNAc most specifically among lectins. By utilizing the high specific binding capacity of the lectin and the glycosylated glycoprotein, the glycoprotein with a specific glycosyl structure can be screened, and in a specific example, the inventor utilizes a self-made AAL 2-agarose gel chromatographic column to enrich the glycoprotein in a serum sample of a healthy individual and/or a patient to obtain a plurality of glycoproteins with terminal N-acetylglucosamine glycosyl.

Those skilled in the art will appreciate that proteins/peptides may be glycosylated to form glycoproteins/glycopeptides, and that glycoproteins/glycopeptides may be deglycosylated to form proteins/peptides. In a glycoprotein in an individual/body fluid/tissue/cell, both the original (non-glycosylated or deglycosylated) and glycosylated forms may exist, and in the case of a glycosylated form, proteins (glycoproteins) often have various forms of existence due to differences in glycosylation sites (sites) and/or extent, i.e., the sugar chains on the glycoprotein have a high degree of macroscopic and microscopic heterogeneity. The glycoproteins Serotransferrin, plasmase C1inhibitor and serpin b4 are also not exceptional, and all three proteins may exist in an individual/body fluid/tissue/cell unglycosylated form, and the respective glycosylated forms all exist in a plurality of different sugar structures, that is, the sugar structure of the same glycoprotein is generally diverse. For example, Serotransferrin may have no sugar groups/chains, or may have one or more sugar chains at one or some sites, with the sugar chain consisting of one or more sugar groups and/or with multiple linkages between sugar groups. The term "glycoprotein having a terminal N-acetylglucosamine sugar group" means that at least one sugar chain of the glycoprotein is terminated with the N-acetylglucosamine sugar group, and the sugar chain includes one or more sugar groups.

It will be understood by those skilled in the art that the three proteins referred to, in the examples, if distinguished by carbohydrate structure, may each be referred to as a class of proteins having one or more carbohydrate structures having a common characteristic of containing at least one terminal N-acetylglucosamine glycosyl group.

In the description which follows, unless otherwise indicated, serum transferrin or Serotransferrin represents serum transferrin having a terminal N-acetylglucosamine group, Plasma protease C1inhibitor or Plasma protease ec 1inhibitor represents Plasma protease C1inhibitor having a terminal N-acetylglucosamine group, and serine protease inhibitor B4 or Serpin B4 represents serine protease inhibitor B4 having a terminal N-acetylglucosamine group.

According to a second aspect of the present invention there is provided the use of a marker according to any one of the above aspects or embodiments in screening for, diagnosing or aiding diagnosis of breast cancer. The foregoing description of the technical features and advantages of the marker in one aspect or any embodiment of the present invention, which is applicable to this aspect of the present invention, will not be repeated herein.

According to an embodiment of the present invention, the screening, diagnosis or diagnosis-assisted breast cancer includes: determining the amount of a marker in the sample to be tested to obtain a first amount, the marker being in any of the aspects or embodiments above; comparing the first amount with a second amount, said second amount being the amount of the corresponding marker in a control sample obtained by the same assay method, and determining the risk of the individual from which the sample is derived for having breast cancer and/or the stage of breast cancer progression based on the comparison obtained. The amount referred to is the amount of protein/glycoprotein expressed, either in absolute or relative amounts, e.g., concentration.

According to an embodiment of the invention, the control sample is a sample of a body fluid selected from at least one of blood, plasma and serum.

The second amount may be measured simultaneously with the first amount when said screening, diagnosis and/or auxiliary diagnosis of breast cancer is performed, or may be recorded or saved as a predetermined measurement. According to the embodiment of the present invention, the amount is a protein concentration, and there is no particular limitation on the selection of the measurement method, and any method capable of quantifying the target protein may be used. Preferably, an assay is selected which is capable of specifically recognizing/binding to the carbohydrate structure of the protein of interest. In a specific example, the assay is selected from at least one of chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, Western Blot (WB), and enzyme-linked immunosorbent assay (ELISA). In one embodiment, the selected assay is enzyme-linked immunosorbent, specifically, enzyme-linked immunosorbent containing lectin, more specifically, lectin is capable of specifically binding to the carbohydrate structure of the glycoprotein in the marker, for example, an antibody can be used to obtain a protein and a complex of the protein (if any), and then the lectin can be used to select a protein with a specific carbohydrate structure for detection. In one specific example, the ELISA used is a Reverse Lectin ELISA (Reverse-Lectin ELISA), more specifically, a Reverse AAL2 Lectin ELISA, and for example, a glycoprotein having a terminal GlcNAc can be specifically bound first with AAL2 Lectin, and then a specific type of glycoprotein can be selected among them with an antibody for detection.

According to an embodiment of the invention, the marker comprises Serotransferrin, and the determination of the Serotransferrin content in the test sample is significantly higher than that in the healthy control sample, as determined by a reverse AAL2 lectin ELISA, the individual from which the test sample is derived is judged to be at high risk of having breast cancer.

According to the embodiment of the invention, the marker comprises a Plasma protease C1inhibitor, the reverse AAL2 lectin ELISA is used for detection, and when the determination result of the content of the Plasma protease C1inhibitor in the sample to be detected is obviously higher than the content of the Plasma protease C1inhibitor in a healthy control sample, the individual from which the sample to be detected comes is judged to have high risk of suffering from breast cancer.

According to the embodiment of the invention, the marker comprises Serpin B4, and when the content of Serpin B4 in the sample to be tested is obviously lower than that in the healthy control sample through detection by reverse AAL2 lectin ELISA, the risk that the individual from which the sample to be tested is from suffering from breast cancer is judged to be high.

According to the embodiment of the present invention, when the marker is a combination protein, the comparison between the detection result of the expression level of any one of the proteins and the healthy control is as described in the above embodiment of the protein, and it is determined that the subject from which the sample to be tested is derived is at high risk of having breast cancer.

According to the embodiment of the present invention, when the marker is a combination protein, the results of the detection of the expression levels of the respective proteins compared with the results of the comparison with the healthy control are as described in the above embodiment of the present invention, and it is determined that the subject from which the sample to be tested is derived is at high risk of having breast cancer.

What is said to be significantly higher or significantly lower can be said to be significantly greater or significantly less, e.g., 5 times, 3 times, 2 times, or more than 1.5 times the greater of the two, or the difference can be statistically significant, e.g., the first amount and the second amount obtained using a measurement method are both a plurality of values, and at a predetermined confidence level, the comparison is made whether the difference between the two sets of values is statistically significant.

According to a third aspect of the present invention, there is provided a kit for use in carrying out the screening, detecting, diagnosing or aiding diagnosis of breast cancer in any one of the above embodiments, the kit comprising an antibody capable of specifically binding to at least one glycoprotein in a marker of any one of the above embodiments, and reagents capable of detecting a complex formed between the glycoprotein and the antibody.

According to embodiments of the invention, the kit further comprises an AAL2 lectin chromatography column, and/or reagents for performing mass spectrometry.

According to a fourth aspect of the present invention, there is provided a method of detecting, screening, diagnosing or aiding diagnosis of breast cancer, the method comprising: determining the amount of the marker in any of the above aspects or any embodiment in the sample to be tested to obtain a first amount; comparing the first amount with a second amount, which is the amount of the corresponding marker in a control sample obtained by the same assay method, and determining the risk of the individual from which the sample is derived from having breast cancer and/or the stage of breast cancer progression based on the comparison. The amount referred to is the amount of protein/glycoprotein expressed, either in absolute or relative amounts, e.g., concentration.

The second amount may be determined at the time of said screening, diagnosis and/or auxiliary diagnosis of breast cancer, or may be pre-determined and recorded or saved. In the examples of the present invention, the amount is a protein concentration, and the concentration is measured by at least one measurement method, and the present invention is not particularly limited with respect to the selection of the measurement method, and any method capable of quantifying the target protein may be used. In a specific example, the assay is selected from at least one of chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, Western Blot (WB), and enzyme-linked immunosorbent assay (ELISA). Preferably, an assay is selected which is capable of specifically recognizing/binding to the carbohydrate structure of the protein of interest. In one embodiment, the selected assay is enzyme-linked immunosorbent, specifically, enzyme-linked immunosorbent containing lectin, more specifically, lectin is capable of specifically binding to the carbohydrate structure of the glycoprotein in the marker, for example, an antibody can be used to obtain a protein and a complex (if any) of the protein, and then the lectin can be used to select a protein with a specific carbohydrate structure for detection. In one specific example, the ELISA used is a Reverse Lectin ELISA (Reverse-Lectin ELISA), more specifically, a Reverse AAL2 Lectin ELISA, and for example, a glycoprotein having a terminal GlcNAc can be specifically bound first with AAL2 Lectin, and then a specific type of glycoprotein can be selected among them with an antibody for detection.

According to the embodiment of the present invention, when the marker is a combination protein, the results of the detection of the expression levels of the respective proteins and the results of the comparison with the healthy control each correspond to the description of the embodiment of the protein above, and it is determined that the subject from which the sample to be tested is derived is at high risk of having breast cancer.

What is said to be significantly higher or significantly lower may be said to be significantly greater or significantly less, e.g. the greater of the two is 5 times, 3 times, 2 times or more than 1.5 times the smaller, or the difference may be statistically significant, e.g. the first and second amounts obtained by a measurement method are both a plurality of values and the comparison of the difference between the two sets of values is statistically significant at a predetermined confidence level.

According to a fifth aspect of the present invention, there is provided a device for detecting, screening, diagnosing or aiding diagnosis of breast cancer, the device being adapted to perform some or all of the steps of the breast cancer detection/screening method of any one of the above aspects or embodiments, the device comprising: a first quantity obtaining module, configured to determine a quantity of the marker in any one of the above embodiments in the sample to be tested, to obtain a first quantity; and an analysis module for comparing the first amount from the first amount obtaining module with a second amount, which is the amount of the corresponding marker in the control sample obtained by the same measuring method, and determining the risk of the individual from which the sample to be measured is derived from having breast cancer and/or the stage of breast cancer development according to the comparison result.

The above description of the technical features and advantages of the breast cancer detection method according to one aspect or any embodiment of the present invention is also applicable to the breast cancer detection apparatus according to this aspect of the present invention, and will not be repeated herein.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram showing the results of detection of the binding specificity of AAL2 saccharide in the examples of the present invention;

FIG. 2 is an electrophoresis diagram of lectin AAL2 affinity chromatography column enriched in each group of glycoproteins in the present example;

FIG. 3 is a mass spectrum of an iTRAQ-LC-MS/MS identification of Serotransferrin in an example of the present invention;

FIG. 4 is a mass spectrum of an iTRAQ-LC-MS/MS identification of Plasma protease C1inhibitor in an example of the present invention;

FIG. 5 is a mass spectrum of iTRAQ-LC-MS/MS identification Serpin B4 in an example of the present invention;

FIG. 6 is a graph showing the results of Western blotting to detect protein expression in examples of the present invention;

FIG. 7 is a diagram showing the results of detecting the expression of Serotransferrin by reverse Lectin-ELISA in the examples of the present invention;

FIG. 8 is a diagram showing the results of the reverse Lectin-ELISA assay for expression of plasmid protease C1inhibitor in the examples of the present invention;

FIG. 9 is a diagram showing the results of detecting the expression of Serpin B4 by reverse Lectin-ELISA in the examples of the present invention.

Detailed Description

The breast cancer markers and the application of the present invention will be described in detail with reference to the following embodiments. The following examples are given for the purpose of illustration only and are not to be construed as limiting the invention. In the description of the present invention, "a plurality" means two or more unless otherwise specified.

Unless otherwise noted, reagents, kits, instruments and software referred to in the following examples are conventional commercially available products or available from open sources, such as agarose gels from eBioscience, inc, iTRAQ kits from courser, inc.

Example 1

The differential expression glycoprotein in the plasma of the breast cancer patient is identified by a method combining lectin affinity chromatography with high performance liquid chromatography/mass spectrometry so as to obtain the breast cancer candidate marker.

1. Sample source and information

Plasma specimens were collected at the sixth national hospital in eastern guan and the young olive hospital in Zhongshan city, and classified as Stage0 according to the seventh edition of the Joint committee for Cancer classification (AJCC) clinical Staging standard for breast Cancer: TisN0M 0; stage I: T1N0M 0; StageII A: T0N1M0, T1N1M0, T2N0M 0: StageIIB: T2N1M0, T3N0M 0; StageIIIA: T0N2M0, T1N2M0, T2N2M0, T3N1-2M 0: stage IIIB: T4N0-2M 0; StageIIIC: any T N3M 0; StageIV: any T any N M1 stage. Wherein Tis is carcinoma in situ, T represents primary tumor, N is regional lymph node, and M represents distant metastasis. Breast cancer specimens are grouped herein according to their sex, age and number of specimens as: control group of healthy women, BC1, BC2, BC 3. The sample data statistics for plasma proteomics analysis are shown in table 1.

TABLE 1

2. Lectin affinity chromatographic column for enriching glycoprotein

Based on previous studies, the lectin AAL2 (agrocyclic egritetin 2) has high binding specificity with a terminal acetylglucosamine glycosyl (terminal GlcNAc), AAL2 is the lectin known to have the highest binding specificity with GlcNAc, and the tail ends of AAL-2 binding glycosyl at the 60-position in the ranking are GlcNAc. FIG. 1 shows the results of the detection of the binding specificity of AAL-2 saccharide by the fifth generation of sugar chip, and it can be seen from FIG. 1 that the sugar structure in the first ten positions of the AAL-2 binding ability is non-reducing GlcNAc at the end; the binding heatmap shows the sugar binding spectrum of AAL-2, and it can be seen that the sugar binding specificity of AAL-2 is very consistent from 0.1. mu.g/ml to 200. mu.g/ml, and that the specific binding capacity to terminal GlcNAc does not vary with the concentration of AAL-2 within this concentration range.

Invention of the inventionBased on the above, human utilizes Sepharose^TMThe 4B-packed adiponectin AAL2 column enriched plasma for terminal acetylglucosamine aminated glycoproteins and it was therefore desirable to be able to screen for early markers in the plasma of breast cancer patients.

The method comprises the following steps of selecting mixed plasma of 8 healthy women and mixed plasma of 8 breast cancer patients (BC1, BC2 and BC3), enriching each group of plasma samples through a lectin AAL2 affinity chromatographic column, and performing primary analysis through SDS-PAGE combined silver staining, wherein the specific operation steps are as follows:

1ml of AAL2Sepharose^TMThe 4B affinity column was washed thoroughly with 5ml of binding buffer at a flow rate of 1ml/min to equilibrate the column environment. The pooled plasma from healthy female control and breast cancer patient groups (8 per group) containing approximately 900. mu.g plasma protein, quantified using the BCA kit, was diluted in 2ml of binding buffer, loaded onto the column and incubated at 4 ℃ for 30min with rotation in a refrigerator. Unbound glycoprotein was washed 3 times with 4ml binding buffer for 10min each. The bound glycoprotein of interest is then eluted 2 times 10min each with 4ml of an eluent (200mM N-acetylglucosamine in binding buffer). Collecting eluates, desalting and concentrating in different ultrafilter tubes, mixing 25 μ l protein concentrate with protein SDS-PAGE sample buffer, heating at 100 deg.C for 10min, loading with equal volume, performing SDS-PAGE, and silver staining.

As a result, a large amount of unbound proteins are washed away by the affinity chromatography column, and several protein bands (especially proteins with molecular weights of 116kD, 80kD, 66.2kD and 40 kD) are enriched in the eluate. Sepharose^TMThe 4B conjugated lectin AAL2 affinity chromatographic column enriches glycoproteins which are specifically bound with AAL2 in mixed plasma of healthy female control group (8 cases) and mixed plasma of breast cancer patient group BC1, BC2 and BC3 (8 cases), respectively, and the enriched glycoproteins are eluted by 3 times of washing. The samples of each step were separated by SDS-PAGE combined silver staining method, as shown in FIG. 2, the four electrophoretograms in FIG. 2a correspond to the mixed plasma of healthy women, BC1, BC2 and BC3, respectively, and the lanes in each electrophoretogram are, from left to right, M: marker; 1: mixing plasma total protein; 2: plasma protein sample breakthrough peak; 3: first, theWashing unbound protein once; 4: washing the unbound protein a second time; 5: washing unbound protein for a third time; 6: competing for the eluted glycoprotein of interest. In FIG. 2b, the lanes are M from left to right: marker 1: glycoproteins of interest eluted from the healthy group; 2: BC1 group eluted glycoprotein of interest; 3: the target glycoproteins eluted from the BC2 group; 4: BC3 group eluted glycoproteins of interest.

Qualitative and quantitative analysis of enriched glycoproteins by i-TRAQ labeling and LC-MS/MS mass spectrometry

Four groups of plasma specimen glycoproteins enriched on lectin affinity chromatography columns were labeled with iTRAQ (isotopic tags for relative and absolute quantification) kit (Health group, iTRAQ 114; BC1 group, iTRAQ 115; BC2 group, iTRAQ 116; BC3, iTRAQ117) and the proteins therein were qualitatively and quantitatively detected by liquid chromatography-secondary mass spectrometry (LC-ESI-MS/MS), wherein a total of 116 proteins were identified.

Specifically, iTRAQ kit labeling was performed according to kit instructions. LC-MS/MS identification was mainly analyzed by combining TripleTOF 5600+ (AB SCIEX, Mississauga, ON) with the split Ultra 1D Plus system (Eksigent, Dublin, CA). The dry peptide fragments screened by SCX were reconstituted in 100. mu.l of Swtichos buffer (0.1% formic acid, 98% ACN), and 20. mu.l of reconstituted sample from each fraction was injected into a nano-LC-MS/MS column spray interface for systematic analysis by a nanospray needle. In series with electrospray time-of-flight mass spectrometry is an online capillary liquid chromatography system. The pooled peptide fragments were eluted in a capillary column of PepMap C-18RP at a flow rate of 300. mu.l/min. The liquid chromatography elution gradient started with 3% buffer b (double distilled water: formic acid: AN ═ 3: 0.1: 97) and 97% buffer a (double distilled water: formic acid: AN ═ 97: 0.1: 3) for 3min, followed by 3% to 25% or 30% buffer b for 60min, followed by 90% buffer b for 7min and finally 3% buffer b for 8 min. When data are collected, the parameters of the machine are set as follows: electrospray positive ionization (ESI)⁺) Detecting, wherein the scanning range is m/z 300-m/z 2000, the temperature of the drying gas is 350 ℃, the flow rate of the drying gas is 10L/min, the pressure of the auxiliary gas is 50psi, and the voltage of the capillary tube is 4000V. Selecting the accumulated time from 2+ to 4+ of the charge quantityFor 3s peptide precursor ions, MALDI-TOP mass spectrometry was automatically modelled to collect MS/MS information for the 5 precursor peaks with the highest intensity.

The inventors excluded interfering proteins such as keratin and the like, and compared to the healthy control group, 59 (> 1.5) of the up-regulated proteins in the BC1 group and 71 of the 12 (< 0.67) of the down-regulated proteins were significantly differentially expressed, including serum transferrin (Serotransferrin) having a terminal N-acetylglucosamine group, Plasma protease C1inhibitor (Plasma protease C1inhibitor) having a terminal N-acetylglucosamine group, and serine protease inhibitor B4(Serpin B4) having a terminal N-acetylglucosamine group, which were subsequently verified. And (3) carrying out protein identification analysis on the screened peptide fragment in a protein group in a database system by using a Pro QUANT software system according to an isotope reporter gene, and simultaneously selecting the protein with the confidence coefficient of more than 95% by using a QUANT ratio Script software system and carrying out quantitative analysis by using m/z115, 116, 117 and 114 to report the peak area integral of ions. And (3) taking m/z114 as a control, selecting a significant difference result (P is less than or equal to 0.05) according to ratios of 115/114, 116/114 and 117/114 for reporting, and selecting a difference protein by taking the ratio of more than 1.50 or less than 0.67 as a threshold value and using the difference protein as a reference range of candidate molecules.

In order to narrow the range of candidate molecules, the inventors selected five proteins for which no glycoprotein reported to be associated with breast cancer and antibodies are commercially available as subjects for further validation by referring to a large amount of literature: serotransferrin, Vitamin K-dependent protein S, Plasma protease C1inhibitor, Serpin B4, C4B-bingprotein beta chain. FIGS. 3, 4 and 5 show the results of mass spectrometry identification of Serostrinia, Plasma protease C1inhibitor and Serpin B4 in these five protein polypeptides. Table 2 shows the information of three proteins that were subsequently validated. The expression of the three proteins has significant difference between tumor patients and healthy people, and compared with the healthy people, the expression of Plasma protease C1inhibitor in the Plasma of the patients is up-regulated, and the expression of Serotransferrin and SerpinB4 is down-regulated. BC1/H represents the ratio of the expression level of the protein in the BC1 group to the expression level in the healthy control group; BC2/H and BC3/H work the same.

TABLE 2

Example 2

The candidate markers were further verified by western blotting.

In order to further verify the expression changes of the three proteins, the inventor detects the expression changes of the proteins in 52 plasma samples of 19 cases of BC1, 9 cases of BC2, 8 cases of BC3, 4 cases of BC4 and 12 cases of healthy people by Western blot. For each set of Western blot experiments, the healthy control H1 was selected as a standard, and the other bands were quantified using WB grayscale analysis software to quantify each protein band by densitometry (densitometry). Results as shown in fig. 6, the expression of the Serotransferrin protein was down-regulated (consistent with mass spectrometry data) in the Stagel group compared to the healthy control group; the expression of the Stage4 group was significantly down-regulated (inconsistent with mass spectrometry data) compared to the healthy control group by Plasma proteaseC1 inhibitor; serpin B4 showed significant downregulation in Stage4 (inconsistent with mass spectral data) compared to healthy controls and Stage1, respectively.

In the above case where the results of mass spectrometry quantification of the contents of these three candidate marker molecules are consistent or inconsistent with the results of Western blot, the inventors believe that the reasons may include: the detection targets for mass spectrometry are AAL2 lectin-enriched glycoproteins that are or are predominantly glycoproteins with terminal GlcNAc, while WB detects glycoproteins directly in plasma samples, including non-glycosylated proteins as well as glycoproteins of various different sugar structures. This inconsistency suggests that a change in the amount of plasmatrose C1inhibitor having a terminal GlcNAc and/or Serpin B4 having a terminal GlcNAc correlates with the occurrence and development of breast cancer, and can serve as a potential breast cancer detection marker.

Example 3

The candidate markers were further verified by Reverse Lectin ELISA (Reverse-Lectin ELISA). The following is the Reverse-Lectin ELISA protocol.

Prepare 4. mu.g/ml AAL2 in 15mmol/l sodium carbonate buffer (ph 9.6) and coat ELISA 96-well plates, 100. mu.l per well, overnight at 4 ℃; the next day, the coating solution is discarded, and PBST is washed once; blocking and incubating with 5% skimmed milk powder blocking solution (5% skimmed milk powder dissolved in PBST solution, pH 7.4) at 300. mu.l per well overnight at 4 deg.C; discarding the confining liquid, adding 300 μ l of washing solution into each well, washing for five times, 3min each time, and patting the plate on filter paper to dry; PBST diluted plasma samples (according to the dilution results of the pre-experiment) by a suitable multiple, 100. mu.l per well, incubated for 1h at room temperature; discard the sample, add 300 μ l PBST wash per well, wash five times for 3min each time, pat the plate dry on filter paper; incubating the target glycoprotein monoclonal antibody, selecting a dilution ratio according to the dilution times of the antibody of a pre-experiment, and incubating for 1h at room temperature; remove primary antibody, add 300 μ l PBST wash per well, wash five times for 3min each time, pat the plate dry on filter paper; HRP streptavidin (1: 1000) was added to the well plate at 100ul per well for incubation for 1 h; removing the enzyme solution, adding 300 μ l of washing solution into each well, washing for five times, 3min each time, and patting the plate dry on filter paper; adding 100 mu l of TMB working solution into each hole, and reacting for 15-30 min; the reaction was stopped with 50ul of stop solution (2mol/L sulfuric acid) and the plate read within 15min at a wavelength of 450 nm.

40 Plasma samples of breast cancer patients, including 19 samples in a BC1 group, 9 samples in a BC2 group, 8 samples in a BC3 group, 4 samples in a BC4 group and 20 samples in healthy female Plasma, were selected, and the expression levels of glycoproteins, Serostrinian, Plasma protease C1inhibitor and Serpin B4 in 60 Plasma samples were measured by a Reversectin ELISA method according to the above-mentioned preliminary experimental conditions.

As shown in fig. 7, expression of glycosylated Serotransferrin (Serotransferrin with terminal GlcNAc) was significantly up-regulated in each of Stage1, Stage2, Stage3, Stage4,. P < 0.05,. P < 0.01, relative to healthy controls.

As shown in fig. 8, expression of glycosylated plasmaarotease C1inhibitor (plasmaarotease C1inhibitor with terminal GlcNAc) was significantly up-regulated in each of

stages

1, 2, 3, 4, P < 0.05, P < 0.01, relative to healthy controls.

As shown in fig. 9, expression of glycosylated Serpin B4(Serpin B4 with terminal GlcNAc) was significantly down-regulated in

stages

1, 2, 3, 4, with P < 0.05 and P < 0.01, relative to healthy controls. The squares and triangles in each figure represent the reverse Lectin-ELISA experimental data for each protein.

In the case that the expression amount changes of the three candidate marker molecules in the disease group and healthy group samples in this example are inconsistent or not completely consistent with the mass spectrometry or Western blot results in example 1 or 2, the inventors believe that the reasons may include: the detection object of mass spectrometry is a glycoprotein enriched with AAL2 lectin and is or mainly a glycoprotein having terminal GlcNAc, while WB directly detects glycoproteins in a plasma sample and is a glycoprotein including non-glycosylated proteins and various sugar structures, and the detection object of ELISA here is a glycoprotein specifically binding to AAL2 lectin and is mainly a glycoprotein having terminal GlcNAc. This inconsistency suggests that changes in the amount of Serotransferrin, Plasma proteaseC 1inhibitor and/or Serpin B4 having terminal GlcNAc are associated with the development of breast cancer and can serve as potential breast cancer detection markers.

Example 4

Plasma samples of any 10 healthy individuals and any 20 breast cancer patients from the previous examples were taken, the information of the individuals from which each sample originated was masked, and the original placement was disrupted, using Serotransferrin (protein one), plasmaprotase C1inhibitor (protein two) and/or Serpin B4 (protein three) protein or protein combination as detection markers, respectively: 1) protein one, 2) protein two, 3) protein three, 4) protein one and protein two, 5) protein one and protein three, 6) protein two and protein three, 7) protein one, protein two and protein three; the following assay comparisons were performed:

the expression level of each protein in the marker in each sample was measured a plurality of times by the method of example 3 (reverse AAL2 lectin ELISA), and a set of measurement values for a certain protein obtained for each sample was compared with the measurement values for the expression level of the protein in the samples of the healthy group of example 3, to determine whether the sample was derived from a breast cancer individual or a healthy individual.

The result shows that the expression quantity of the protein or the protein combination 1) to 7) is detected to detect the breast cancer, and the method can be used for breast cancer diagnosis or auxiliary diagnosis. Specifically, the detection rate of each of the single proteins 1) -3) is higher than 80%, and the classification accuracy is not less than 70%; and 4) protein combination type 4) to 7), and the detection rate is not less than 55% and the classification accuracy is more than 92% when various proteins exist and the detection and judgment results are not conflicted.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A breast cancer marker comprising serum transferrin having a terminal N-acetylglucosamine glycosyl and a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl, wherein the marker is capable of specifically binding to agaricus bisporus lectin 2.

2. The marker of claim 1, characterized in that said marker further comprises serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl group.

3. The marker according to claim 1 or 2, characterized in that said terminal N-acetylglucosamine glycosyl is a non-reduced terminal N-acetylglucosamine glycosyl.

4. A kit for diagnosing or aiding in the diagnosis of breast cancer, the kit comprising an antibody capable of specifically binding to a protein in a marker according to any one of claims 1 to 3, and a reagent capable of detecting a complex formed by the protein and the antibody, the kit comprising agrocybe aegerita lectin 2.

5. An apparatus for detecting breast cancer, comprising:

a first quantity obtaining module for determining the quantity of the marker of any one of claims 1 to 3 in a sample to be tested to obtain a first quantity;

and an analysis module for comparing the first amount from the first amount obtaining module with a second amount, which is the amount of the corresponding marker in the control sample obtained by the same measuring method, and determining the risk of the individual from which the sample to be measured is derived from having breast cancer and/or the period of the breast cancer development according to the comparison result.

6. The device of claim 5, wherein the control sample is from a healthy individual.

7. The device of claim 5, wherein said control sample is from a breast cancer patient.

8. The device of claim 5, wherein the control sample is a sample of a bodily fluid selected from at least one of blood, plasma, and serum.

9. The device according to any of claims 5-8, characterized in that the determination is performed using at least one determination method.

10. The device of claim 9, wherein said assay is selected from at least one of chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, western blotting, and enzyme-linked immunosorbent assay.