CN107782897B - Markers for breast cancer - Google Patents

Abstract

The invention relates to the field of biomedical detection and provides a breast cancer marker. Said marker includes at least one of the following glycoproteins: serum transferrin with terminal N-acetylglucosamine glycosyl, plasma proteinase C1 inhibitory factor with terminal N-acetylglucosamine glycosyl, and plasma proteinase C1 inhibitor with terminal N-acetylglucosamine glycosyl Acetylglucosamine glycosyl serine protease inhibitor B4. Detecting the amount of any one, two or all three of the three proteins in the sample to be tested can determine or assist in determining the occurrence and/or development of breast cancer. These several kinds of proteins can be used alone or in any combination as markers for breast cancer screening or auxiliary screening and diagnosis.

Description

breast cancer markers

technical field

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

Background technique

Breast cancer is a malignant tumor that occurs in the glandular epithelium of the breast. The global incidence of breast cancer has been increasing since the late 1970s. According to the incidence data of breast cancer released by the National Cancer Center and the Bureau of Disease Control and Prevention of the Ministry of Health in 2012, the incidence of breast cancer in the national cancer registration area ranks first among female malignant tumors.

According to the seventh edition of the breast cancer clinical staging standards of the American Joint Commission for Cancer Staging (AJCC), breast cancer can be divided into Stage0: TisN0M0; Stage I: T1N0M0; Stage IIA: T0N1M0, T1N1M0, T2N0M0; StageIIB: T2N1M0, T3N0M0; StageIIIA: T0N2M0, T1N2M0, T2N2M0, T3N1-2M0; StageIIIB: T4N0-2M0; StageIIIC: any TN3M0; StageIV: any T any N M1stage. Among them, Tis refers to carcinoma in situ, T refers to primary tumor, N refers to regional lymph node, and M refers to distant metastasis.

Breast cancer in situ is not fatal; however, because breast cancer cells lose the properties of normal cells, the connections between cells are loose and easily sloughed off. Once the cancer cells fall off, the free cancer cells can spread throughout the body with the blood or lymph, forming metastasis and life-threatening.

Breast cancer screening and monitoring, especially early screening, auxiliary diagnosis, and diagnosis, are of great significance for the early detection and follow-up judgment of breast cancer, and can improve the success rate of curing breast cancer.

SUMMARY OF THE INVENTION

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

According to the first aspect of the present invention, the present invention provides a breast cancer marker, the marker includes at least one of the following glycoproteins: serum transferrin (Serotransferrin) with terminal N-acetylglucosamine sugar group , a plasma protease C1 inhibitor (Plasma protease C1 inhibitor) with a terminal N-acetylglucosamine glycosyl and a serine protease inhibitor B4 (Serpin B4) with a terminal N-acetylglucosamine glycosyl.

The above three glycoproteins with specific glycosyl structures are the result of the inventors using Agrocybe agerita lectin 2 (AAL2), which was isolated and isolated by the research team earlier, and using lectin affinity chromatography combined with high performance liquid phase. The method of chromatography/mass spectrometry was used to identify the differentially expressed glycoproteins in the plasma of breast cancer patients, which was verified by further screening and experiments. The inventors found that these three glycoproteins with specific glycosyl structures are differentially expressed in breast cancer patients and even breast cancer patients at different stages, and detect any one, two or all of these three proteins in the sample to be tested. The three concentrations can judge or assist in judging the occurrence and/or development of breast cancer, and these determined proteins alone or in any combination can be used as markers for breast cancer screening or auxiliary screening and diagnosis.

According to an embodiment of the present invention, the marker is a glycoprotein. In a specific example, the marker is serum transferrin with a terminal N-acetylglucosamine sugar group. In another specific example, the marker is a plasma proteinase C1 inhibitor with a terminal N-acetylglucosamine glycosyl group. In yet another specific example, the marker is serpin B4 with a terminal N-acetylglucosamine glycosyl group.

According to an embodiment of the present invention, the so-called marker is a composition, which is composed of multiple glycoproteins. In one specific example, the marker is composed of serum transferrin with a terminal N-acetylglucosamine saccharide and plasma protease C1 inhibitor with a terminal N-acetylglucosamine saccharide. In a specific example, the marker is a protein composition consisting of serum transferrin with a terminal N-acetylglucosamine glycosyl group and serpin B4 with a terminal N-acetylglucosamine glycosyl group. In a specific example, the marker is a protein composition consisting of plasma proteinase C1 inhibitor with terminal N-acetylglucosamine glycosyl and serine protease inhibitor B4 with terminal N-acetylglucosamine glycosyl. In a specific example, the marker is a protein composition consisting of serum transferrin with a terminal N-acetylglucosamine glycosyl, plasma protease C1 inhibitor with a terminal N-acetylglucosamine glycosyl, and a terminal N-acetylglucosamine glycosyl N-acetylglucosamine glycosyl-based serine protease inhibitor B4 composition.

According to an embodiment of the present invention, the terminal N-acetylglucosamine sugar group possessed by the marker is a non-reducing terminal N-acetylglucosamine sugar group.

According to an embodiment of the present invention, the so-called marker can specifically bind to the lectin AAL2. Glycochip experiments of AAL2 showed that the highest binding specificity of AAL2 is the terminal N-acetylglucosamine (GlcNAc) [Jiang S, Chen Y, Wang M, et al. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine [J]. The Biochemical Journal, 2012, 443(2): 369-378], the specificity of AAL2 for GlcNAc is higher than that of other lectins that bind to GlcNAc, and it is the only known lectin that binds to GlcNAc. tallest. Using the high specific binding ability of the lectin to glycosylated glycoproteins, glycoproteins with specific glycosyl structures can be screened. In a specific example, the inventor used a self-made AAL2-Sepharose column. Glycoproteins in serum samples of healthy individuals and/or patients are enriched to obtain a variety of glycoproteins with terminal N-acetylglucosamine glycosyl groups.

Those skilled in the art can know that a protein/peptide can be converted into a glycoprotein/glycopeptide after glycosylation, and a glycoprotein/glycopeptide can be converted into a protein/peptide after deglycosylation. A glycoprotein in an individual/fluid/tissue/cell that may exist in both native (non-glycosylated or deglycosylated) and glycosylated forms, and, for glycosylated form proteins (glycoproteins) Often due to different positions (sites) and/or degrees of glycosylation, there are multiple forms of existence, that is, sugar chains on glycoproteins have a high degree of macroscopic and microscopic heterogeneity. The so-called glycoproteins Serotransferrin, Plasma protease C1 inhibitor and SerpinB4 are no exception. In individuals/humors/tissues/cells, these three proteins may all have non-glycosylated forms, and their respective glycosylated forms exist. A variety of different sugar structures, that is, the sugar structures of the same glycoprotein are generally diverse. For example, Serotransferrin may have no glycans/glycans, or it may have one or more glycans at one or some positions, and the glycans consist of one or more glycans and/or glycans There are many ways to connect. The so-called glycoprotein with terminal N-acetylglucosamine sugar group refers to that the end of at least one sugar chain of the glycoprotein is an N-acetylglucosamine sugar group, and the so-called sugar chain includes one or more sugar groups.

Those skilled in the art can understand that, in the example, if the three proteins are distinguished by their sugar structures, each of them can be referred to as a class of proteins, which are proteins with one or more sugar structures. A common feature of carbohydrate structures is the inclusion of at least one terminal N-acetylglucosamine sugar group.

In the following description of this article, unless otherwise specified, serum transferrin or Serotransferrin means serum transferrin with terminal N-acetylglucosamine glycosyl, plasma protease C1 inhibitor or Plasma proteaseC1 inhibitor means terminal N-acetylglucosamine Glycosylated plasma protease C1 inhibitor, serpin B4 or Serpin B4 means serpin B4 with a terminal N-acetylglucosamine glycosyl.

According to a second aspect of the present invention, there is provided the use of the marker in the above-mentioned aspect or any one of the embodiments in screening, diagnosing or assisting in the 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 is still applicable to the use of this aspect of the present invention, and will not be repeated here.

According to an embodiment of the present invention, the so-called screening, diagnosis or auxiliary diagnosis of breast cancer includes: determining the amount of the marker in the sample to be tested, and obtaining the first amount, and the marker is any one of the above-mentioned aspects or any of the embodiments. comparing the first amount and the second amount, and according to the obtained comparison results, determine the risk of breast cancer and/or the development stage of breast cancer in the source individual of the sample to be tested, the second amount refers to the use of the same Determine the amount of the corresponding marker in the control sample obtained by the method. The said amount is the expression amount of protein/glycoprotein, which can be absolute content or relative content, such as concentration.

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

The so-called second amount may be determined simultaneously with the first amount during the screening, diagnosis and/or auxiliary diagnosis of breast cancer, or may be pre-determined and recorded or saved. According to the embodiment of the present invention, the amount is the protein concentration, and the selection of the measurement method is not particularly limited, and any method that can quantify the target protein is acceptable. Preferably, an assay method that can specifically recognize/bind to the carbohydrate structure of the target protein is selected. In a specific example, the assay method is selected from at least one of chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, Western Blot (WB) and enzyme-linked immunosorbent assay (ELISA). In a specific example, the selected assay method is enzyme-linked immunosorbent assay, specifically, the so-called enzyme-linked immunosorbent assay is an enzyme-linked immunosorbent assay comprising a lectin, more specifically, the lectin here is one that can be combined with a marker For example, an antibody can be used to obtain a certain protein and its complex (if any), and then a lectin can be used to select a protein with a specific carbohydrate structure for detection. . In a specific example, the ELISA used is a Reverse-Lectin ELISA, more specifically, a Reverse-Lectin ELISA, for example, AAL2 lectin can be used to specifically bind a sugar having a terminal GlcNAc first proteins, and then use antibodies to select specific classes of glycoproteins for detection.

According to an embodiment of the present invention, the marker includes Serotransferrin, which is detected by reverse AAL2 lectin ELISA. When the measurement result of the content of Serotransferrin in the sample to be tested is significantly higher than its content in the healthy control sample, it is determined that the sample to be tested is from of individuals are at high risk for breast cancer.

According to an embodiment of the present invention, the marker includes Plasma protease C1 inhibitor, which is detected by reverse AAL2 lectin ELISA, and the determination result of the content of Plasma protease C1 inhibitor in the sample to be tested is significantly higher than its content in the healthy control sample, The individual from whom the sample to be tested is determined to be at high risk for breast cancer.

According to an embodiment of the present invention, the marker includes Serpin B4, which is detected by reverse AAL2 lectin ELISA. When the measurement result of the Serpin B4 content in the sample to be tested is significantly lower than its content in the healthy control sample, the determination to be tested is determined. The sample is from an individual who is at high risk for breast cancer.

According to an embodiment of the present invention, when the marker is a combination protein, the comparison result of any protein expression detection result and the healthy control conforms to the description of the above-mentioned protein embodiment, and it is determined that the individual from which the sample to be tested is derived has the risk of breast cancer high.

According to the embodiment of the present invention, when the marker is a combination protein, the comparison results of the detection results of various proteins and the healthy controls are in line with the description of the above-mentioned embodiment of the protein, and it is determined that the individual from which the sample to be tested is derived has breast cancer. High risk.

The term “significantly higher” or “significantly lower” may mean “significantly greater” or “significantly less than” in general, for example, the larger of the two is 5 times, 3 times, 2 times, or 1.5 times or more of the smaller. It can be that the difference is statistically significant, for example, the first quantity and the second quantity obtained by an assay method are multiple values, and at a predetermined confidence level, whether the difference between the two groups of values is statistically significant .

According to a third aspect of the present invention, there is provided a kit for implementing the purposes of screening, detecting, diagnosing or assisting in the diagnosis of breast cancer in any of the above-mentioned embodiments, the kit includes a kit that can be combined with any of the above-mentioned methods. Antibodies that specifically bind to at least one glycoprotein among the markers in the examples, and reagents that can be used to detect complexes formed by the glycoprotein and said antibody.

According to an embodiment of the present invention, the kit further includes 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 for detecting, screening, diagnosing or assisting in diagnosing breast cancer, the method comprising: determining the amount of the marker in any of the above-mentioned aspects or any of the embodiments in the sample to be tested , obtain the first amount; compare the first amount with the second amount, the second amount is the amount of the corresponding marker in the control sample obtained by the same assay method, and according to the comparison result, determine the amount of the sample to be tested. The source individual's risk of having breast cancer and/or the stage of breast cancer development. The said amount is the expression amount of protein/glycoprotein, which can be absolute content or relative content, such as concentration.

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

The so-called second amount may be determined during the screening, diagnosis and/or auxiliary diagnosis of breast cancer, or may be pre-determined and recorded or saved. In the embodiments of the present invention, the amount is the protein concentration, and at least one assay method is used to measure the concentration. The present invention has no particular limitation on the selection of the assay method, and any method that can quantify the target protein is acceptable. Preferably, an assay method that can specifically recognize/bind to the carbohydrate structure of the target protein is selected. In a specific example, the assay method is selected from at least one of chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, Western Blot (WB) and enzyme-linked immunosorbent assay (ELISA). In a specific example, the selected assay method is enzyme-linked immunosorbent assay, specifically, the so-called enzyme-linked immunosorbent assay is an enzyme-linked immunosorbent assay comprising a lectin, more specifically, the lectin here is one that can be combined with a marker For example, an antibody can be used to obtain a certain protein and its complex (if any), and then a lectin can be used to select a protein with a specific carbohydrate structure for detection. . In a specific example, the ELISA used is a Reverse-Lectin ELISA, more specifically, a Reverse-Lectin ELISA, for example, AAL2 lectin can be used to specifically bind a sugar having a terminal GlcNAc first proteins, and then use antibodies to select specific classes of glycoproteins for detection. According to an embodiment of the present invention, the marker includes Serotransferrin, which is detected by reverse AAL2 lectin ELISA. When the measurement result of the content of Serotransferrin in the sample to be tested is significantly higher than its content in the healthy control sample, it is determined that the sample to be tested is from of individuals are at high risk for breast cancer.

According to an embodiment of the present invention, the marker includes Plasma protease C1 inhibitor, which is detected by reverse AAL2 lectin ELISA, and the determination result of the content of Plasma protease C1 inhibitor in the sample to be tested is significantly higher than its content in the healthy control sample , it is determined that the individual from which the test sample is derived has a high risk of breast cancer.

According to an embodiment of the present invention, the marker includes Serpin B4, which is detected by reverse AAL2 lectin ELISA. When the measurement result of the Serpin B4 content in the sample to be tested is significantly lower than its content in the healthy control sample, the determination to be tested is determined. The sample is from an individual who is at high risk for breast cancer.

According to an embodiment of the present invention, when the marker is a combination protein, the comparison result of any protein expression detection result and the healthy control conforms to the description of the above-mentioned protein embodiment, and it is determined that the individual from which the sample to be tested is derived has the risk of breast cancer high.

According to the embodiment of the present invention, when the marker is a combination protein, the comparison results of the detection results of various protein expression levels and the healthy controls are in line with the description of the above-mentioned embodiment of the protein, and it is determined that the individual from which the sample to be tested is derived has breast cancer high risk.

The so-called significantly higher or significantly lower can be generally said to be significantly larger or significantly smaller, for example, the larger value of the two is 5 times, 3 times, 2 times or 1.5 times or more of the smaller value, or It can be that the difference is statistically significant, for example, the first quantity and the second quantity obtained by an assay method are multiple values, and at a predetermined confidence level, whether the difference between the two groups of values is statistically significant .

According to a fifth aspect of the present invention, there is provided a device for detecting, screening, diagnosing or assisting in diagnosing breast cancer, the device being used to implement any of the above aspects or part of the breast cancer detection/screening method in any of the embodiments step or all steps, the device includes: a first quantity acquisition module, which is used to measure the quantity of the marker in any of the above-mentioned embodiments in the sample to be tested, and obtain the first quantity; an analysis module, which is used for Comparing the first amount and the second amount from the first amount acquisition module, the second amount is the amount of the corresponding marker in the control sample obtained by the same assay method, and according to the comparison result, determine the source of the sample to be tested The individual's risk of having breast cancer and/or the period of breast cancer development.

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

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

Description of drawings

Fig. 1 is the schematic diagram of the detection result of AAL2 sugar binding specificity in the embodiment of the present invention;

Fig. 2 is the electrophoretogram of each group of glycoproteins enriched by the lectin AAL2 affinity chromatography column in the embodiment of the present invention;

Fig. 3 is the mass spectrogram of identifying Serotransferrin by iTRAQ-LC-MS/MS in the embodiment of the present invention;

Fig. 4 is the mass spectrum of iTRAQ-LC-MS/MS identifying Plasma protease C1 inhibitor in the embodiment of the present invention;

Fig. 5 is the mass spectrogram of iTRAQ-LC-MS/MS identifying SerpinB4 in the embodiment of the present invention;

FIG. 6 is a schematic diagram of the results of detecting protein expression by western blotting in the embodiment of the present invention;

7 is a schematic diagram of the results of reverse Lectin-ELISA detection of Serotransferrin expression in the embodiment of the present invention;

FIG. 8 is a schematic diagram of the results of detecting the expression of Plasmaprotease C1 inhibitor by reverse Lectin-ELISA in the embodiment of the present invention;

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

Detailed ways

The breast cancer markers and applications of the present invention will be described in detail below with reference to specific embodiments. The following examples are only used to explain the present invention and should not be construed as a limitation of the present invention. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

Unless otherwise stated, the reagents, kits, instruments and software that are not specifically described in the following examples are conventional commercially available products or open source, such as agarose gels purchased from eBioscience in the United States, and iTRAQ kits purchased from Huijun Biological Company, etc.

Example 1

Lectin affinity chromatography combined with high performance liquid chromatography/mass spectrometry was used to identify differentially expressed glycoproteins in the plasma of breast cancer patients, in order to obtain candidate markers of breast cancer.

1. Sample source and information

Plasma specimens were collected in Dongguan Sixth People's Hospital and Zhongshan Xiaolan Hospital. According to the seventh edition of the breast cancer clinical staging standard of the American Joint Commission for Cancer Staging (AJCC), they were divided into Stage0: TisNOM0; Stage I : T1N0M0; Stage IIA: T0N1M0, T1N1M0, T2NOM0; StageIIB: T2N1M0, T3NOM0; Stage IIIA: T0N2M0, T1N2M0, T2N2M0, T3N1-2M0; Stage IIIB: T4N0-2M0; Stage IIIC: any T N3 M0; Stage IV: any T any N Mlstage. Among them, Tis is carcinoma in situ, T stands for primary tumor, N stands for regional lymph node, and M stands for distant metastasis. The breast cancer specimens were grouped here according to their gender, age and number of specimens: healthy female control group, BC1, BC2, BC3. The sample data statistics for plasma proteomic analysis are shown in Table 1.

Table 1

2. Lectin Affinity Chromatography Column for Enrichment of Glycoproteins

Based on previous studies, the lectin AAL2 (Agrocybe agerita lectin 2) has a high binding specificity to the terminal acetylglucosamine glycosyl (terminal GlcNAc), and AAL2 has the highest binding specificity to GlcNAc among the known lectins. AAL-2 binding ranks The ends of the first 60 glycosyl groups are all GlcNAc. Figure 1 shows the results of the fifth-generation sugar chip to detect the binding specificity of AAL-2 sugars. From Figure 1, it can be seen that the top ten glycosyl structures of AAL-2 binding ability have non-reducing GlcNAc ends; the binding heat map The glycosyl-binding profile of AAL-2 is shown, and it can be seen that the glycosyl-binding specificity of AAL-2 is very consistent from 0.1 μg/ml to 200 μg/ml, and in this concentration range, its specific binding ability to terminal GlcNAc is not will vary with the concentration of AAL-2.

Based on the above, the inventors used Sepharose ^TM 4B packing Eurolectin AAL2 chromatography column to enrich the terminal acetylglucosaminated glycoproteins in plasma, and then hope to be able to screen early markers in the plasma of breast cancer patients.

Pooled plasma from 8 healthy women and 8 patients with breast cancer in each group (BC1, BC2, BC3) were selected. The plasma samples of each group were enriched by lectin AAL2 affinity chromatography column and combined by SDS-PAGE. The silver staining was initially analyzed, and the specific operation steps were as follows:

1 ml of AAL2Sepharose ^™ 4B affinity chromatography column was thoroughly rinsed with 5 ml of binding buffer at a flow rate of 1 ml/min to equilibrate the internal environment of the column. Quantified by BCA kit, the mixed plasma of healthy female control group and breast cancer patient samples containing about 900μg plasma protein (8 people in each group) was diluted in 2ml of binding buffer, loaded on the column, and stored in a refrigerator at 4°C Incubate with rotation for 30 min. Unbound glycoproteins were washed 3 times with 4 ml of binding buffer for 10 min each. Then, the bound target glycoprotein was eluted with 4 ml of eluent (200 mM N-acetylglucosamine dissolved in the binding buffer), eluted twice, 10 min each time. The eluates of each group were collected and placed in different ultrafiltration tubes for desalting and concentration. 25 μl of protein concentrate was mixed with protein SDS-PAGE loading buffer, heated at 100 °C for 10 min, and the same volume was loaded for SDS-PAGE and silver. dye.

The results showed that a large number of unbound proteins were washed away through the affinity chromatography column, and several protein bands were enriched in the eluate (especially proteins with molecular weights of 116kD, 80kD, 66.2kD, and 40kD). Sepharose ^TM 4B-coupled lectin AAL2 affinity chromatography column was used to enrich the pooled plasma of healthy women control group (8 cases) and breast cancer patient group BC1, BC2, BC3 pooled plasma (8 cases each) that specifically bind to AAL2. Glycoproteins, respectively, were washed three times, and the enriched glycoproteins were finally eluted. The samples at each step were separated by SDS-PAGE combined with silver staining, as shown in Figure 2. The four electropherograms in Figure 2a correspond to the mixed plasma of healthy women, BC1, BC2, and BC3, respectively. The lanes in each electropherogram are from the left. From right to right, M: marker; 1: mixed plasma total protein; 2: plasma protein sample breakthrough peak; 3: unbound protein in the first wash; 4: unbound protein in the second wash; 5: third Unbound protein was washed twice; 6: Competitive elution of target glycoprotein. In Figure 2b, the lanes are M: marker 1: target glycoprotein eluted from healthy group; 2: target glycoprotein eluted from BC1 group; 3: target glycoprotein eluted from BC2 group; 4: BC3 group eluted glycoprotein of interest.

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

Glycoproteins from four groups of plasma samples enriched by lectin affinity chromatography were labeled with iTRAQ (isobaric tags for relative and absolute quantitation) kits (Health group, iTRAQ114; BC1 group, iTRAQ115; BC2 group, iTRAQ116; BC3, iTRAQ117), the proteins were qualitatively and quantitatively detected by liquid chromatography-secondary mass spectrometry (LC-ESI-MS/MS), and a total of 116 proteins were identified.

Specifically, iTRAQ kit labeling was performed according to the kit instructions. LC-MS/MS identification was mainly performed using the combined analysis of the TripleTOF 5600+ (AB SCIEX, Mississauga, ON) and the splitless Ultra 1D Plus system (Eksigent, Dublin, CA). The dried peptides screened by SCX were reconstituted in 100 μl of Swtichos buffer (0.1% formic acid, 98% ACN), and the reconstituted sample of 20 μl of each component was injected into the nano-LC-MS/ MS column spray interface for system analysis. In tandem with electrospray time-of-flight mass spectrometry is an online capillary liquid chromatography system. The mixed peptides were eluted and separated in a PepMap C-18 RP capillary column at a flow rate of 300 μl/min. The LC elution gradient started with 3% BufferB (double distilled water:formic acid:AN=3:0.1:97) and 97% BufferA (double distilled water:formic acid:AN=97:0.1:3) for 3 min , and then eluted with 3%~25% or 30% BufferB for 60min, 90% BufferB for 7min, and finally with 3% BufferB for 8min. During data collection, the parameters of the machine are set as follows: electrospray positive ionization (ESI ⁺ ) detection, the scanning range is m/z 300～m/z 2000, the drying gas temperature is 350℃, the drying gas flow rate is 10L/min, the auxiliary gas pressure 50psi, capillary voltage 4000V. The peptide precursor ions with the charge from 2+ to 4+ accumulated for 3s were selected, and the MALDI-TOP mass spectrometer automatically adopted the mode to collect the MS/MS information of the five precursor peaks with the highest intensity.

The inventors excluded interfering proteins such as keratin. Compared with the healthy control group, there were 59 up-regulated proteins (>1.5) and 12 down-regulated proteins (<0.67) in the BC1 group, among which there were significantly differentially expressed proteins. There are 71, including subsequent validated serum transferrin (Serotransferrin) with terminal N-acetylglucosamine glycosyl, plasma protease C1 inhibitor with terminal N-acetylglucosamine glycosyl (Plasma protease C1 inhibitor) and A terminal N-acetylglucosamine glycosyl serine protease inhibitor B4 (Serpin B4). Use the Pro QUANT software system to perform protein identification analysis on the screened peptides according to the isotopic reporter gene to the proteome in its database system. The Quant Ration Script software system selects proteins with a confidence of more than 95% and uses m/z115, 116, 117 Quantitative analysis was performed by integrating the peak area of 114 reporter ions. With m/z114 as the control, according to the ratio of 115/114, 116/114, 117/114, select the significant difference results (P≤0.05) to report, with the ratio >1.50 or <0.67 as the threshold value to select the differential protein, and use it as the threshold. Reference range for candidate molecules.

In order to narrow down the range of candidate molecules, the inventors reviewed a large number of documents and selected five proteins for which no reported glycoproteins related to breast cancer were found and antibodies were available for further verification: Serotransferrin, Vitamin K-dependent protein S, Plasma protease C1inhibitor, Serpin B4, C4b-bingprotein beta chain. Figure 3, Figure 4 and Figure 5 show the results of Serotransferrin, Plasmaprotease C1 inhibitor and Serpin B4 among the five protein polypeptides identified by mass spectrometry. Table 2 shows information on the three proteins that were subsequently validated. The expression of these three proteins is significantly different between tumor patients and healthy people. Compared with healthy people, Plasma protease C1 inhibitor in plasma of patients is up-regulated, while Serotransferrin and SerpinB4 are down-regulated. BC1/H represents the ratio of protein expression in the BC1 group to that in the healthy control group; the same is true for BC2/H and BC3/H.

Table 2

Example 2

The above candidate markers were further verified by western blotting.

To further verify the expression changes of these three proteins, the inventors detected the protein expression changes in 52 plasma samples from 19 BC1, 9 BC2, 8 BC3, 4 BC4 and 12 healthy people by Western blot. For each set of Western blot experiments, the healthy control H1 was selected as the standard, the other bands were quantified by WB grayscale analysis software, and each protein band was quantified by densitometry. The results are shown in Figure 6. Compared with the healthy control group, the expression of Serotransferrin protein was down-regulated in the Stage1 group (consistent with the mass spectrometry data); compared with the healthy control group, the expression of PlasmaproteaseC1 inhibitor in the Stage4 group was significantly down-regulated (inconsistent with the mass spectrometry data). ; SerpinB4 was significantly down-regulated in Stage4 group compared with healthy control group and Stage4 group respectively (inconsistent with mass spectrometry data).

The above-mentioned mass spectrometry quantitative results of the content of these three candidate marker molecules are consistent or inconsistent with the Western blot results. The inventor believes that the reasons may include: the detection object of mass spectrometry is the glycoprotein enriched by AAL2 lectin, which is or It is mainly glycoproteins with terminal GlcNAc, while WB directly detects glycoproteins in plasma samples, including non-glycosylated proteins and glycoproteins with various glycostructures. This inconsistency implies that the changes in the amount of Plasmaprotease C1 inhibitor with terminal GlcNAc and/or Serpin B4 with terminal GlcNAc are related to the occurrence and development of breast cancer, and can be used as potential breast cancer detection markers.

Example 3

The above candidate markers were further validated by Reverse-Lectin ELISA. The following is the reverse-Lectin ELISA test flow.

Prepare 4μg/ml AAL2 in 15mmol/l sodium carbonate buffer solution (ph 9.6), coat ELISA 96-well plate, 100μl per well, overnight at 4°C; the next day, discard the coating solution, wash once with PBST; % nonfat milk powder blocking solution (5% nonfat milk powder dissolved in PBST solution, pH 7.4) 300 μl per well for blocking and incubation at 4°C overnight; discard the blocking solution, add 300 μl washing solution to each well, wash five times, 3 min each time, Pat the plate dry on filter paper; dilute the appropriate multiples of plasma samples with PBST (according to the pre-experiment dilution results), add 100 μl to each well, and incubate at room temperature for 1 h; discard the samples, add 300 μl of PBST washing solution to each well, and wash five times, each time 3 min, pat the plate dry on filter paper; incubate the target glycoprotein monoclonal antibody, select the dilution ratio according to the antibody dilution ratio of the pre-experiment, and incubate at room temperature for 1 h; remove the primary antibody, add 300 μl of PBST washing solution to each well, and wash five times , 3min each time, pat the plate dry on the filter paper; 100ul of HRP streptavidin (1:1000) per well was added to the well plate and incubated for 1 h; the enzyme solution was removed, and 300 μl of washing solution was added to each well, and washed five times. After 3 minutes, the plate was patted dry on filter paper; 100 μl of TMB working solution per well was added to the plate, and the reaction was performed for 15 to 30 minutes; 50 μl of stop solution (2mol/L sulfuric acid) was used to stop the reaction, and the plate was read at a wavelength of 450 nm within 15 minutes.

40 plasma samples from breast cancer patients were selected, including 19 patients in BC1 group, 9 patients in BC2 group, 8 patients in BC3 group, 4 patients in BC4 group and 20 plasma samples from healthy women. Experimental conditions, the expression levels of glycoproteins Serotransferrin, Plasma protease C1inhibitor and Serpin B4 in these 60 plasma samples were detected by Reverselectin ELISA method.

As shown in Figure 7, the expression of glycosylated Serotransferrin (Serotransferrin with terminal GlcNAc) was significantly up-regulated in Stage1, Stage2, Stage3, and Stage4 groups relative to the healthy control group, **P<0.05, ***P <0.01.

As shown in Figure 8, relative to the healthy control group, the expression of glycosylated Plasma protease C1 inhibitor (Plasma protease C1 inhibitor with terminal GlcNAc) was significantly up-regulated in Stagel, Stage2, Stage3, Stage4 groups, **P<0.05 , ***P<0.01.

As shown in Figure 9, the expression of glycosylated Serpin B4 (SerpinB4 with terminal GlcNAc) was significantly down-regulated in each of Stagel, Stage2, Stage3, and Stage4 groups relative to the healthy control group, **P<0.05, *** P<0.01. The square dots and triangle dots in each figure represent the reverse Lectin-ELISA experimental data of each protein.

In the case where the expression 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 inventor believes that the reasons may include: The detection objects of mass spectrometry are glycoproteins enriched by AAL2 lectin, or mainly glycoproteins with terminal GlcNAc, while WB directly detects glycoproteins in plasma samples, including non-glycosylated proteins and various sugars. The structure of the glycoprotein, and the detection object of the ELISA here is the glycoprotein that specifically binds to the AAL2 lectin, mainly the glycoprotein with terminal GlcNAc. This inconsistency suggests that changes in the amount of Serotransferrin with terminal GlcNAc, Plasma proteaseC1 inhibitor and/or Serpin B4 with terminal GlcNAc are related to the occurrence and development of breast cancer, and can be used as potential breast cancer detection markers.

Example 4

Take the plasma samples of any 10 healthy individuals and any 20 breast cancer patients in the previous example, mask the information of each sample source individual and disrupt the original placement position, use Serotransferrin (protein one), Plasmaprotease Clinhibitor (protein two) respectively. ) and/or Serpin B4 (protein three) protein or protein combination as detection markers: 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; carry out the following assay comparisons:

Referring to the method of Example 3 (reverse AAL2 lectin ELISA), the expression of each protein in the markers in each sample was detected multiple times, and a set of detection values of a certain protein obtained from each sample was compared with Example 3. The detection value of the protein expression level in the healthy group samples is compared to determine whether the sample is from a breast cancer individual or a healthy individual.

The results show that detecting the expression levels of proteins or protein combinations 1)-7) to detect breast cancer can be used for breast cancer diagnosis or auxiliary diagnosis. Specifically, the detection rates of single proteins 1)-3) are all higher than 80%, and the classification accuracy rates are not less than 70%; protein combination types 4)-7), all kinds of proteins exist and the detection results are inconsistent Conflicts are counted, the detection rate is not less than 55%, and the classification correct rate is more than 92%.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms 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.

Although embodiments of the present 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 in these embodiments without departing from the principles and spirit of the invention, The scope of the invention is defined by the claims and their equivalents.

Claims (10)

1. A breast cancer marker comprising a plasma protease C1inhibitor having a terminal N-acetylglucosamine glycosyl and a serine protease inhibitor B4 having a terminal N-acetylglucosamine glycosyl, said marker being capable of specifically binding to agaricus bisporus lectin 2.

2. The marker of claim 1, characterized in that it further comprises serum transferrin 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 use in the assisted 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 reagents for detecting a complex formed between the protein and the antibody, the kit comprising a chromatography column for 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 the analysis module is used for comparing the first quantity from the first quantity acquisition module with a second quantity, and judging the risk that the source individual of the sample to be detected has the breast cancer according to the comparison result, wherein the second quantity is the quantity of the corresponding marker in the control sample obtained by using the same determination method.

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 of any of claims 5-8, wherein said measurement is performed using at least one measurement 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.

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