阅读说明：本技术 用于检测大肠癌的生物标志物 (Biomarker for detecting colorectal cancer ) 是由 朝长毅 白水崇 于 2018-06-29 设计创作，主要内容包括：提供一种用于早期检测大肠癌的生物标志物。一种用于检测大肠癌的大肠癌生物标志物,其由下面的1至22的22个蛋白质中的至少一个蛋白质或者1至22的蛋白质的部分肽中的至少一个肽构成：1.膜联蛋白A11；2.膜联蛋白A3；3.膜联蛋白A4；4.腱生蛋白-N；5.转铁蛋白受体蛋白1；6.葡萄糖转运蛋白1；7.补体成分C9；8.CD88抗原；9.78kDa葡萄糖调节蛋白；10.α-1-酸性糖蛋白；11.基质金属蛋白酶9；12.血管生成素-1；13.CD67抗原；14.粘蛋白-5B；15.衔接蛋白GRB2；16.膜联蛋白A5；17.嗅觉介导素-4；18.中性氨基酸转运蛋白B(0)；19.三肽基肽酶1；20.热休克相关70kDa蛋白2；21.蛋白酶体亚基α型-5；以及22.中性粒细胞明胶酶相关脂质运载蛋白。(A biomarker for early detection of colon cancer is provided, which comprises at least one of the following 22 proteins 1 to 22 or at least one of partial peptides of the proteins 1 to 22, 1-annexin A11, 2-annexin A3, 3-annexin A4, 4-tenascin-N, 5-transferrin receptor protein 1, 6-glucose transporter 1, 7-complement component C9, 8-CD 88 antigen, 9.78kDa glucose regulatory protein, 10- α -1-acid glycoprotein, 11-matrix metalloproteinase 9, 12-angiopoietin-1, 13-CD 67 antigen, 14-mucin-5B, 15-GRB 2, 16-adaptor annexin A5, 17-olfactory mediated protein-4, 18-neutral amino acid transporter B (0), 19-based peptidase 1, 20-heat-related protein α, and gelatin-related protein subunit.)

1. A biomarker for detecting colon cancer, comprising at least one of the following 22 proteins or at least one peptide selected from partial peptides of the 1 to 22 proteins:

1. annexin a11(ANXA 11) represented by sequence No. 1;

2. annexin a3(ANXA 3) represented by sequence No. 2;

3. annexin a4(ANXA 4) represented by sequence No. 3;

4. tenascin n (tnn) represented by sequence No. 4;

5. transferrin receptor protein 1(TFRC) represented by sequence number 5;

6. GLUT-1(SLC2A1) which is a glucose transporter-1 represented by SEQ ID NO. 6;

7. complement component C9(C9) represented by sequence No. 7;

8.CD88 antigen (C5AR1) represented by sequence No. 8;

9. a 78kDa glucose regulating protein (HSPA5) represented by seq id No. 9;

10.α -1-acid glycoprotein (ORM1) represented by SEQ ID NO. 10;

11. matrix metalloproteinase 9(MMP9) represented by sequence No. 11;

12. angiopoietin-1 (ANGPT1) represented by sequence number 12;

13.CD67 antigen represented by sequence number 13 (CEACAM 8);

14. mucin-5B (MUC5B) represented by sequence number 14;

15. the adaptor protein GRB2(GRB2) represented by SEQ ID NO. 15;

16. annexin a5(ANXA 5) represented by sequence No. 16;

17. olfactin-4 (OLFM4) represented by sequence number 17;

18. neutral amino acid transporter B (0) (SLC1a5) represented by seq id No. 18;

19. tripeptidyl peptidase 1(TPP1) represented by sequence No. 19;

20. heat shock associated 70kDa protein 2(HSPA2) represented by sequence number 20;

21. proteasome subunit α type-5 (PSMA5) represented by SEQ ID NO. 21, or

22. Neutrophil gelatinase-associated lipocalin (LCN2) represented by sequence number 22.

2. The biomarker for detecting colon cancer according to claim 1,

the biomarker for colorectal cancer is a combination of two or more of the 22 proteins according to claim 1 to 22 or two or more of the partial peptides of the proteins 1 to 22.

3. The biomarker for detecting colon cancer according to claim 1 or 2,

the partial peptide of the protein according to claim 1 to 22 is a peptide consisting of the amino acid sequence represented by SEQ ID NO. 23 to 59.

4. The biomarker for detecting colon cancer according to claim 1,

the biomarker of colorectal cancer is composed of at least one of the following 12 proteins or at least one peptide of partial peptides of the 12 proteins:

1. annexin a11 represented by sequence No. 1;

2. annexin a3 represented by sequence No. 2;

3. annexin a4 represented by sequence No. 3;

4. tenascin-N represented by sequence No. 4;

5. transferrin receptor protein 1 represented by sequence number 5;

6. glucose transporter-1 represented by sequence No. 6;

8.CD88 antigen represented by sequence number 8;

11. matrix metalloproteinase 9 represented by sequence No. 11;

16. annexin a5 represented by sequence number 16;

17. olfactin-4 represented by sequence number 17;

19. tripeptidyl peptidase 1 represented by SEQ ID NO. 19; or

22. Neutrophil gelatinase-associated lipocalin represented by sequence number 22.

5. The biomarker for detecting colon cancer according to claim 4,

the biomarker for colorectal cancer is a combination of 2 or more proteins among the 12 proteins according to claim 4 or 2 or more peptides among partial peptides of the 12 proteins.

6. The biomarker for detecting colon cancer according to claim 4 or 5,

the partial peptide of 12 proteins according to claim 4, which is a peptide consisting of the amino acid sequence represented by SEQ ID Nos. 23 to 34, 37, 38, 43, 44, 53, 54, 56 and 59.

7. The biomarker for detecting colon cancer according to claim 1,

the biomarker of the colorectal cancer is composed of annexin A4 or annexin A11 or partial peptides of the annexin A4 or the annexin A11.

8. The biomarker for detecting colon cancer according to claim 7,

the biomarker for colorectal cancer is formed by combining annexin A4 and annexin A11 or combining a partial peptide of annexin A4 and a partial peptide of annexin A11.

9. The biomarker for detecting colon cancer according to claim 7 or 8,

the partial peptides of annexin a4 and annexin a11 are peptides consisting of the amino acid sequences represented by seq id nos 23, 24, 27 and 28.

10. A biomarker for detecting colon cancer, comprising the combination of the biomarker according to any of claims 1 to 9 and CEA.

11. A method for detecting colon cancer, which comprises measuring the biomarker for colon cancer according to any one of claims 1 to 10 in a biological sample.

12. A method for detecting colon cancer, characterized in that a biomarker for colon cancer according to any one of claims 1 to 10 in a biological sample is measured, and when the biomarker is present at a higher concentration than in a healthy normal person, it is judged that colon cancer has occurred.

13. The method for detecting colon cancer according to claim 11 or 12,

the biological sample is extracellular vesicles EV in blood.

14. The method of detecting colon cancer according to any one of claims 11 to 13,

detection is by immunological assays or mass spectrometry.

15. A kit for detecting colon cancer, comprising an antibody against the biomarker for colon cancer according to any one of claims 1 to 10 in a biological sample.

Technical Field

The present invention relates to a novel protein or peptide biomarker which can be used for detecting colon cancer.

Background

Colorectal cancer (CRC) is one of the most frequent cancers in the world, and development of effective biomarkers for CRC is indispensable for improving human survival rate. However, the diagnostic methods for colorectal cancer used at present are all diagnostic methods with very low accuracy such as fecal occult blood and CEA, and no new biomarker capable of diagnosing colorectal cancer at an early stage with high accuracy has been found, and there is a strong demand for a blood biomarker.

To date, despite the discovery of multiple cancer-associated factors and cancer biomarker candidates through large-scale omics studies, there are no blood biomarkers that have reached clinical use. As evidence for this, few biomarkers recognized by the U.S. Food and Drug Administration (FDA) in recent years are under two per year. It is generally believed that one of the reasons for this stagnation is that there are serious problems in the strategy of biomarker development.

Conventionally, in many clinical examinations, detection of protein biomarkers is performed by antibody-based quantitative analysis (mainly using enzyme-linked antibody immunoassay (ELISA)). These methods greatly depend on the quality of the antibody, so that precision, particularly specificity, is problematic, and it is difficult to simultaneously evaluate multiple items of marker candidates.

Due to recent advances in Mass Spectrometry (MS), the possibility of significant changes in clinical screening has emerged. This is because the selective reaction monitoring/multiple reaction monitoring (SRM/MRM), which is a representative method of targeted proteomics, can quantify not only biomarker proteins with high accuracy but also a plurality of items of markers simultaneously. In the previous reports, Whiteaker et al used the SRM/MRM method to quantify candidate proteins for multiple breast cancer biomarkers in patient plasma (see non-patent document 1). In addition, some examples of biomarker candidate proteins for various cancers verified by the SRM/MRM method have been reported, including the report of the problem group by the present inventors (see non-patent documents 2 to 4).

Therefore, targeted proteomics using SRM/MRM methods is a powerful tool for finding biomarkers.

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a biomarker for early detection of colorectal cancer.

Means for solving the problems

The present inventors first searched for a CRC biomarker candidate protein from a literature search of PubMed. To detect these biomarker candidate proteins in blood, Extracellular Vesicles (EV) in blood were noted. This is because, in addition to the fact that EV has recently attracted attention as a mediator for intercellular communication, EV is considered to be involved in the onset and progression of various diseases and is considered to include many biomarker candidates. In addition, when a minute amount of protein in blood is to be detected using a mass spectrometer, detection of a minute amount of a candidate protein for a marker may be inhibited due to inclusion of a large amount of protein in blood such as albumin. However, when purifying EV in blood, a large amount of contaminating proteins is removed, and thus a trace amount of biomarker candidate proteins in EV can be detected. Thus, EV-containing proteins are considered promising biomarker candidates.

The present inventors have found a biomarker candidate protein that is a candidate for protein reduction that may have a possibility of EV existing in blood by a literature search, and have quantified these proteins by using the SRM/MRM method. As a result thereof, some biomarker candidates for early diagnosis of CRC were determined, and the present invention was completed.

Namely, the present invention is as follows:

[1] a biomarker for detecting colon cancer, which is composed of at least one of the following 22 proteins or at least one peptide of partial peptides of the proteins 1 to 22:

1. annexin a11(Annexin a11) (ANXA 11) (seq id No. 1);

2. annexin A3(Annexin A3) (ANXA 3) (seq id No. 2);

3. annexin a4(Annexin a4) (ANXA 4) (seq id No. 3);

4. tenascin-N (Tanascin-N) (TNN) (seq id No. 4);

5. transferrin receptor protein 1(TFRC) (sequence No. 5);

6. glucose transporter-1 (GLUT-1) (SLC2A1) (SEQ ID NO: 6);

7. complement component C9 (complementary component C9) (C9) (SEQ ID NO: 7);

CD88 antigen (CD88 antigen) (C5AR1) (SEQ ID NO: 8);

9.78kDa glucose-regulated protein (78kDa glucose-regulated protein) (HSPA5) (SEQ ID NO: 9);

α -1-acid glycoprotein (α -1-acid glycoprotein) (ORM1) (SEQ ID NO. 10);

11. matrix metalloproteinase 9(Matrix metalloproteinase-9) (MMP9) (seq id No. 11);

12. angiopoietin-1 (Angiopoietin-1) (ANGPT1) (seq id No. 12);

CD67 antigen (CD67 antigen) (CEACAM8) (seq id No. 13);

14. Mucin-5B (Mucin-5B) (MUC5B) (SEQ ID NO: 14);

15. the adaptor protein GRB2(Adapter protein GRB2) (GRB2) (SEQ ID NO: 15);

16. annexin a5(Annexin a5) (ANXA 5) (seq id No. 16);

17. olfactomedin-4 (olfmedin-4) (OLFM4) (seq id No. 17);

18. neutral amino acid transporter B (0) (Neutral amino acid transporter B (0)) (SLC1A5) (SEQ ID NO: 18);

19. tripeptidyl-peptidase-1 (TPP1) (SEQ ID NO: 19);

20. heat shock associated 70kDa protein 2(Heat shock-related 70kDa protein 2) (HSPA2) (SEQ ID NO: 20);

21. proteasome subunit α type-5 (Proteasome subunit subBunit alpha type-5) (PSMA5) (SEQ ID NO: 21), or

22. Neutrophil gelatinase-associated lipocalin (LCN2) (SEQ ID NO: 22).

[2] The biomarker for detecting colon cancer according to [1], wherein two or more of the 22 proteins of 1 to 22 of [1] or two or more of the partial peptides of the proteins of 1 to 22 are combined.

[3] The biomarker for detecting colon cancer according to [1] or [2], wherein the partial peptide of the protein 1 to 22 of [1] is a peptide having an amino acid sequence represented by SEQ ID Nos. 23 to 59.

[4] The biomarker for detecting colon cancer according to [1], which comprises at least one of the following 12 proteins or at least one peptide of partial peptides of the 12 proteins:

1. annexin a11 (seq id No. 1);

2. annexin a3 (seq id No. 2);

3. annexin a4 (seq id No. 3);

4. tenascin-N (SEQ ID NO: 4);

5. transferrin receptor protein 1 (SEQ ID NO: 5);

6. glucose transporter-1 (SEQ ID NO: 6);

CD88 antigen (SEQ ID NO: 8);

11. matrix metalloproteinase 9 (SEQ ID NO: 11);

16. annexin a5 (seq id No. 16);

17. olfactin-4 (SEQ ID NO: 17);

19. tripeptidyl peptidase 1 (SEQ ID NO: 19); or

22. Neutrophil gelatinase-associated lipocalin (SEQ ID NO: 22)

[5] The biomarker for detecting colon cancer according to [4], wherein 2 or more proteins out of 12 proteins of [4] or 2 or more peptides out of partial peptides of the 12 proteins are combined.

[6] The biomarker for detecting colon cancer according to [4] or [5], wherein the partial peptide of the 12 proteins of [4] is a peptide having an amino acid sequence represented by SEQ ID Nos. 23 to 34, 37, 38, 43, 44, 53, 54, 56 and 59.

[7] The biomarker for detecting colon cancer according to [1], wherein the biomarker for colon cancer is composed of annexin A4 or annexin A11 or partial peptides thereof.

[8] The biomarker for detecting colon cancer according to [7], wherein the biomarker is a combination of annexin A4 and annexin A11 or a combination of a partial peptide of annexin A4 and a partial peptide of annexin A11.

[9] The biomarker for detecting colon cancer according to [7] or [8], wherein the partial peptides of annexin A4 and annexin A11 are peptides consisting of the amino acid sequences represented by SEQ ID Nos. 23, 24, 27 and 28.

[10] A colon cancer biomarker for detecting colon cancer, which is a combination of the colon cancer biomarker according to any one of [1] to [9] and CEA.

[11] A method for detecting colon cancer, which comprises measuring a biomarker for colon cancer according to any one of [1] to [10] in a biological sample.

[12] A method for detecting colon cancer, which comprises measuring a biomarker for colon cancer according to any one of [1] to [10] in a biological sample, and determining that colon cancer is present at a higher concentration than in a healthy normal person.

[13] The method for detecting colon cancer according to [11] or [12], wherein the biological sample is Extracellular Vesicles (EV) in blood.

[14] The method for detecting colon cancer according to any one of [11] to [13], wherein the detection is carried out by an immunological assay method or a mass spectrometry method.

[15] A colorectal cancer detection kit comprising an antibody against any of the colorectal cancer biomarkers [1] to [10] in a biological sample.

The present specification contains the disclosure of japanese patent application No. 2017-129941, which is the basis of the priority of the present application.

ADVANTAGEOUS EFFECTS OF INVENTION

The biomarker of the present invention can detect colorectal cancer, particularly early colorectal cancer, with high sensitivity and high specificity. In addition, by combining the biomarkers, detection can be performed with further high sensitivity and high specificity.

Drawings

Fig. 1 is a diagram showing strategies for exploring large intestine cancer markers in Extracellular Vesicles (EVs). The number of candidate proteins selected in each step is shown in the figure.

Fig. 2 is a diagram showing a method for shotgun proteomic analysis of CRC biomarker candidate proteins in EV. FIG. 2a shows the steps of EV production and MS analysis, and FIG. 2b shows the results of Venturi map analysis of biomarker candidate proteins identified by shotgun proteome analysis and EV proteins. Of the EV proteins identified from serum or cell culture supernatant, 356 colorectal cancer biomarker candidate proteins were identified as EV proteins.

FIG. 3 is a diagram showing SRM target protein (46) and peptide (71).

FIG. 4 is a graph showing a list of proteins validated with a high degree of accuracy (AUC > 0.7).

Fig. 5-1 is a graph showing the results of relative quantification of peptides in serum between three sets (N, C and Cm) based on SRM analysis. N indicates non-cancer control patients, C indicates cancer patients without metastasis, and Cm indicates cancer patients with metastasis. The dot plots represent the peak area ratio of endogenous to SI peptides (p <0.05, p <0.01, N.S indicates no significant difference). The vertical axis represents the Area Ratio.

Fig. 5-2 is a graph showing the results of relative quantification of peptides between three sets (N, C and Cm) based on SRM analysis (continuation of fig. 5-1).

Fig. 5-3 is a graph showing the results of relative quantification of peptides between three sets (N, C and Cm) based on SRM analysis (continuation of fig. 5-2).

Fig. 5-4 is a graph showing the results of relative quantification of peptides between three sets (N, C and Cm) based on SRM analysis (continuation of fig. 5-3).

Fig. 5-5 are graphs (continuation of fig. 5-4) showing the results of relative quantitation of peptides between three sets (N, C and Cm) based on SRM analysis.

Fig. 5-6 are graphs (continuation of fig. 5-5) showing the results of relative quantitation of peptides between three sets (N, C and Cm) based on SRM analysis.

FIG. 6-1 is a graph (one) showing the results of statistical analysis of a target peptide. Fig. 6-1a shows the results of relative quantification of peptides between three sets (N, C and Cm) based on SRM analysis. N indicates a non-cancer control, C indicates cancer without metastasis, and Cm indicates cancer with metastasis. The dot plots represent the peak area ratio of endogenous to SI peptides (p <0.05, p <0.01, N.S indicates no significant difference). The vertical axis represents the Area Ratio. FIG. 6-1b shows the results of ROC curve analysis for identifying N and C (1) and for identifying C and Cm (2). The area under the curve (AUC) used for identification is shown in the respective graphs. The ordinate represents sensitivity (sensitivity), and the abscissa represents 1-specificity (specificity).

Fig. 6-2 is a graph showing the results of statistical analysis of the target peptide (second). Fig. 6-2c shows the results of relative quantification of peptides between three sets (N, C and Cm) based on SRM analysis. N indicates a non-cancer control, C indicates cancer without metastasis, and Cm indicates cancer with metastasis. The dot plots represent the peak area ratio of endogenous to SI peptides (p <0.05, p <0.01, N.S indicates no significant difference). The vertical axis represents the Area Ratio. Fig. 6-2d show the results of ROC curve analysis for identifying N and C (1) and for identifying C and Cm (2). The area under the curve (AUC) used for identification is shown in the respective graphs. The ordinate represents sensitivity (sensitivity), and the abscissa represents 1-specificity (specificity).

Fig. 7-1 is (one of) a graph showing an ROC curve (1) for identifying N and C and an ROC curve (2) for identifying C and Cm. The area under the curve (AUC) used for identification is shown in the respective graphs. The ordinate represents sensitivity (sensitivity), and the abscissa represents 1-specificity (specificity).

FIG. 7-2 is a diagram (continuation of FIG. 7-1) showing an ROC curve (1) for identifying N and C and an ROC curve (2) for identifying C and Cm.

Fig. 7-3 are diagrams (continuation of fig. 7-2) showing the ROC curve (1) for identifying N and C and the ROC curve (2) for identifying C and Cm.

Fig. 7-4 are diagrams (continuation of fig. 7-3) showing the ROC curve (1) for identifying N and C and the ROC curve (2) for identifying C and Cm.

Fig. 7-5 are diagrams (continuation of fig. 7-4) showing the ROC curve (1) for identifying N and C and the ROC curve (2) for identifying C and Cm.

Fig. 7-6 are diagrams (continuation of fig. 7-5) showing the ROC curve (1) for identifying N and C and the ROC curve (2) for identifying C and Cm.

FIG. 8-1 is a graph (one of) showing the results of ROC curve analysis of combinations of target peptides. The sensitivity of the diagnosis when the peptides were combined was evaluated between N and C. The area under the curve (AUC), sensitivity (sensitivity), and specificity (specificity) are shown in the respective graphs.

Fig. 8-2 is a graph showing the results of ROC curve analysis of the combination of target peptides (second). The sensitivity of the diagnosis when the peptides were combined was evaluated between N and C. The area under the curve (AUC), sensitivity (sensitivity), and specificity (specificity) are shown in the respective graphs.

Fig. 9 is a graph showing the results of ROC curve analysis of combinations of target peptides. The sensitivity of the diagnosis when the peptides were combined was evaluated between N and C. The area under the curve (AUC), sensitivity (sensitivity), and specificity (specificity) are shown in the respective graphs. FIG. 9 shows the combination of higher accuracy (AUC > 0.9). Other combinations are shown in FIGS. 8-1 and 8-2.

FIG. 10-1 is a graph showing the result of comparing the sensitivity (sensitivity) of the target peptide to CEA. The sensitivity when the cutoff value was set to the maximum peak area of N was calculated as the ratio of the samples (the broken line indicates 100% specificity). The vertical axis represents the Area Ratio.

FIG. 10-2 is a graph showing the result of comparison of sensitivity (sensitivity) of the target peptide to CEA (continuation of FIG. 10-1).

FIG. 10-3 is a graph showing the result of comparison of sensitivity (sensitivity) of the target peptide to CEA (continuation of FIG. 10-2).

FIG. 11 is a graph showing the results of comparing the sensitivity of the target peptide to CEA. The sensitivity (sensitivity) when the Cutoff value (Cutoff) was set to the maximum peak area of N was calculated as the ratio of the samples (the dotted line indicates 100% specificity). The three peptides with the highest sensitivity in recognition of N and C are shown in fig. 11. The vertical axis of the graph with respect to the three peptides represents Area Ratio. Other peptides with higher sensitivity than CEA are shown in FIGS. 10-1-10-3.

Detailed Description

The present invention will be described in detail below.

The present invention relates to a protein or a partial peptide thereof which is a biomarker for detecting colon cancer (CRC). The present invention also provides a method for detecting colon cancer using the biomarker. Further, the present invention is a method for obtaining auxiliary data for diagnosing colorectal cancer using the biomarker. By using the biomarker of the present invention, colorectal cancer can be detected at an early stage.

Biomarkers for use in the present invention can be explored by, for example, proteomic analysis techniques. For example, the following steps can be used to search for the target.

First, proteins that are candidates for biomarkers were searched for by quantitative shotgun proteomics using tissues of colorectal cancer patients. In this case, a protein showing a change between tissues can be targeted as a candidate protein by comparing and quantifying the large intestine tissue or benign tumor tissue of the large intestine, the tissue of a case of large intestine cancer without metastasis, and the tissue of a case of large intestine cancer with metastasis. In addition, the biomarker candidate protein can also be searched for by using a protein that has been considered to be involved in the onset and progression of colorectal cancer in the conventional studies. The examination of the change in expression level between tissues can be performed by a targeted proteomics method using the SRM/MRM (Selected interaction monitoring)/Multiple interaction monitoring) method (Gillette MA et al, Nat methods.2013; 10: 28-34) which is a method of selectively destroying parent ions of specific masses for these biomarker candidates and detecting specific ions in the generated daughter ions, thereby enabling the target protein-derived peptides to be detected with high sensitivity from within a complicated sample And (4) quantifying.

In the method of the present invention, the following proteins are listed as biomarkers for detecting colon cancer:

1. annexin a11(Annexin a11) (ANXA 11) (seq id No. 1);

2. annexin A3(Annexin A3) (ANXA 3) (seq id No. 2);

3. annexin a4(Annexin a4) (ANXA 4) (seq id No. 3);

4. tenascin-N (Tanascin-N) (TNN) (seq id No. 4);

5. transferrin receptor protein 1(TFRC) (sequence No. 5);

6. glucose transporter-1 (GLUT-1) (SLC2A1) (SEQ ID NO: 6);

7. complement component C9 (complementary component C9) (C9) (SEQ ID NO: 7);

CD88 antigen (CD88 antigen) (C5AR1) (SEQ ID NO: 8);

9.78kDa glucose-regulated protein (78kDa glucose-regulated protein) (HSPA5) (SEQ ID NO: 9);

α -1-acid glycoprotein (α -1-acid glycoprotein) (ORM1) (SEQ ID NO. 10);

11. matrix metalloproteinase 9(Matrix metalloproteinase-9) (MMP9) (seq id No. 11);

12. angiopoietin-1 (Angiopoietin-1) (ANGPT1) (seq id No. 12);

CD67 antigen (CD67 antigen) (CEACAM8) (seq id No. 13);

14. Mucin-5B (Mucin-5B) (MUC5B) (SEQ ID NO: 14);

15. adapter protein GRB2(Adapter protein GRB2) (GRB2) (SEQ ID NO. 15)

16. Annexin a5(Annexin a5) (ANXA 5) (seq id No. 16);

17. olfactomedin-4 (olfmedin-4) (OLFM4) (seq id No. 17);

18. neutral amino acid transporter B (0) (Neutral amino acid transporter B (0)) (SLC1A5) (SEQ ID NO: 18);

19. tripeptidyl-peptidase 1(TPP1) (SEQ ID NO: 19);

20. heat shock associated 70kDa protein 2(Heat shock-related 70kDa protein 2) (HSPA2) (SEQ ID NO: 20);

21. proteasome subunit α type-5 (Proteasome subunit subBunit alpha type-5) (PSMA5) (SEQ ID NO: 21), or

22. Neutrophil gelatinase-associated lipocalin (LCN2) (SEQ ID NO: 22).

In addition, as the partial peptide of the above protein which can be used as a biomarker for detecting colon cancer, there can be mentioned:

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 23 or 24 (partial peptide of annexin A11),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 25 or 26 (partial peptide of annexin A3),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 27 or 28 (partial peptide of annexin A4),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 29 or 30 (partial peptide of tenascin-N),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 31 or 32 (a partial peptide of transferrin receptor protein 1),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 33 or 34 (partial peptide of glucose transporter-1),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 35 or 36 (partial peptide of complement component C9),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 37 or 38 (partial peptide of CD88 antigen),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 39 or 40 (partial peptide of 78kDa glucose regulating protein),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 41 or 42 (α -1-acid glycoprotein partial peptide),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 43 or 44 (partial peptide of matrix metalloproteinase 9)

A peptide consisting of the amino acid sequence represented by SEQ ID NO. 45 or 46 (a partial peptide of angiopoietin-1),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 47 or 48 (partial peptide of CD67 antigen),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 49 or 50 (partial peptide of mucin-5B),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 51 or 52 (partial peptide of adaptor protein GRB2),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 53 (partial peptide of annexin A5);

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 54 (partial peptide of olfactory mediator-4),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 55 (partial peptide of neutral amino acid transporter-B (0)),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 56 (partial peptide of tripeptidyl peptidase 1),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 57 (partial peptide of heat shock-associated 70kDa protein 2),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 58 (partial peptide of proteasome subunit α type-5),

a peptide consisting of the amino acid sequence represented by SEQ ID NO. 59 (a partial peptide of neutrophil gelatinase-associated lipocalin), and the like.

In the present invention, the above-mentioned protein or a partial peptide of the protein is used as a biomarker, but the protein or peptide includes a protein or peptide having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequences represented by SEQ ID Nos. 1 to 59, and these proteins or peptides can also be used as a biomarker in the method of the present invention. As used herein, "one or more" means "one or three", "one or two", or "one".

The peptide generally refers to a substance having a molecular weight of 1 ten thousand or less in which amino acids are linked by peptide bonds, and the number of amino acid residues is several to 50 or less. In the present specification, a protein or a partial peptide thereof may be used as a biomarker, and when it is referred to as a partial peptide, the following peptides are referred to: the partial amino acid sequence having a part of the amino acid sequence of the protein is not limited, but the molecular weight is preferably 1 ten thousand or less. The partial peptide may be produced in the course of expression synthesis by transcription and translation, or may be produced as a digestion degradation product peptide after being synthesized into a protein and digested and degraded in vivo.

Among these proteins and peptides, the following proteins and peptides are preferably used as biomarkers for detection of colon cancer: the results of ROC analysis have revealed that glucose transporter-1, transferrin receptor protein 1, annexin a5, matrix metalloproteinase 9, annexin a4, annexin a11, tenascin-N, annexin A3, olfactory mediator-4, CD88 antigen, neutrophil gelatinase-associated lipocalin or tripeptidyl peptidase 1, or partial peptides thereof, which are responsible for colorectal cancer, can be detected with good detection sensitivity and specificity. Further preferably, annexin a4 or annexin a11 or partial peptides thereof are used as biomarkers for detecting colorectal cancer.

In the present invention, at least one protein and/or partial peptide among the above 22 proteins and partial peptides can be used as the biomarker, but 2 or more, more preferably 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, or 22 proteins or partial peptides are preferably used, and thus colorectal cancer can be detected with higher accuracy. In this case, a combination of different proteins may be used, or a combination of a certain protein and a partial peptide or a partial peptide of a protein other than the protein may be used. In addition, only a plurality of intact proteins may be used as a biomarker. In this way, by using a plurality of proteins or partial peptides as biomarkers, colon cancer can be detected more accurately, and the state of progression of colon cancer can be determined accurately.

Examples thereof include: combination of glucose transporter-1 and transferrin receptor protein 1, combination of glucose transporter-1 and angiopoietin-1, combination of matrix metalloproteinase 9 and transferrin receptor protein 1, combination of transferrin receptor protein 1 and neutrophil gelatinase-associated lipocalin, combination of transferrin receptor protein 1 and angiopoietin 1, combination of transferrin receptor protein 1 and adaptor protein, combination of 78kDa glucose regulator protein and tripeptide peptidase 1, combination of tenascin N and neutrophil gelatinase-associated lipocalin, combination of transferrin receptor protein 1 and neutrophil gelatinase-associated lipocalin and angiopoietin-1, combination of transferrin receptor protein 1 and olfactory mediator-4, combination of tenascin-N and angiopoietin-1, and combinations of these, Combinations of olfactory mediator-4 with angiopoietin-1, tenascin-N with olfactory mediator-4, olfactory mediator-4 with tripeptidyl peptidase 1. Or a combination of partial peptides of the respective proteins.

In addition, by using the above-described protein or partial peptide in combination with CEA (carcinoembryonic antigen), which has been known as a biomarker for colorectal cancer in the past, as a biomarker for detecting colorectal cancer, detection can be performed with higher sensitivity and higher specificity than that by using the protein or partial peptide alone.

In patients with colorectal cancer, the expression of the above protein or partial peptide is increased. Therefore, it is sufficient to quantify these proteins or partial peptides in blood. Here, blood includes serum and plasma. In addition, the above proteins are present in Extracellular Vesicles (EV). Therefore, it is possible to detect proteins or partial peptides in EV by separating EV present in blood and concentrating it if necessary. EV can be separated into EV fractions by, for example, ultracentrifugation. In addition, separation can be performed by EV specific markers. Markers for EV include CD9, CD63, CD81, and the like, and can be separated using magnetic beads or the like to which antibodies against these markers are bound. In addition, Phosphatidylserine (PS) is an example of another EV marker, and can be separated using magnetic beads to which phosphatidylserine binding proteins having affinity for phosphatidylserine are bound. Examples of the phosphatidylserine binding protein include: molecule 4 containing the immunoglobulin/mucin domain of T cells (Tim4), Annexin a5(Annexin a5), Annexin v (Annexin v), milk fat globule-EGF factor protein 8(MFG-E8), and the like.

In the method of the present invention, a protein/partial peptide map in a biological sample is examined, but the biological sample used is not limited. Examples of the liquid sample include liquid samples that can be collected from a living body, such as whole blood, serum, plasma, urine, saliva, and other body fluids. In addition, since proteins, partial proteins, and partial peptides in the biological sample may be further decomposed depending on the storage state of the biological sample, the biological sample is preferably used without repeating freezing and thawing. In addition, a protease inhibitor or the like may be added to the biological sample. Alternatively, it is also possible to isolate exosomes using FACS (fluorescence activated cell sorter) or flow cytometer using the above markers as indicators.

Extracting protein or peptide from the isolated exosome, and measuring the protein or peptide.

Proteins or partial peptides can be detected by various methods. The following are examples, but are not limited to these methods.

For example, the measurement can be performed by an immunological measurement method using an antibody against each protein or partial peptide to be detected. Examples of the immunological measurement methods include a solid-phase Immunoassay (RIA, EIA, FIA, CLIA, etc.), a dot blot method, a Latex Agglutination method (LA: Latex Agglutination-Turbidimetric Immunoassay), and an immunochromatography. The antibody can be immobilized on a substrate.

Among them, from the viewpoint of quantitativeness, an ELISA (Enzyme-Linked ImmunoSorbent Assay) method, which is one of EIA (Enzyme Immunoassay) methods, is preferable. In the ELISA method, a sample is added to a well of a microtiter plate in which an antibody is immobilized, and an antigen-antibody reaction is performed on the sample, and an enzyme-labeled antibody is further added to perform an antigen-antibody reaction on the sample, and after washing, the sample is reacted with an enzyme substrate and developed, and the absorbance is measured to detect a marker protein or a partial peptide in the sample, and the concentration of the protein or the partial peptide in the sample can be calculated from the measured value. Further, fluorescence may be measured by subjecting them to an antigen-antibody reaction using a fluorescent-labeled antibody. The antigen-antibody reaction may be carried out at 4 to 45 ℃, more preferably at 20 to 40 ℃, still more preferably at 25 to 38 ℃, and the reaction time may be about 10 minutes to 18 hours, more preferably about 10 minutes to 1 hour, still more preferably about 30 minutes to 1 hour.

The antibody against the marker protein or partial peptide used in the immunological method may be a monoclonal antibody, a polyclonal antibody, or Fab, F (ab') of a monoclonal antibody₂And the like bind to the active fragment.

The present invention also encompasses a test reagent or a kit for detecting colon cancer, which comprises an antibody against one or more of the above 22 proteins or partial peptides thereof.

Biomarker proteins for detecting colon cancer or partial peptides thereof can also be analyzed by mass spectrometry using a mass spectrometer. Mass spectrometry is particularly useful for the detection of partial peptides.

Alternatively, the detection may be performed using a mass spectrometer. In particular for the detection of partial peptides of biomarker proteins, the use of mass spectrometers is suitable. The mass spectrometer includes a sample introduction unit, an ionization chamber, an analysis unit, a detection unit, a recording unit, and the like. As the ionization method, an electron impact ionization (EI) method, a Chemical Ionization (CI) method, a Field Desorption (FD) method, a Secondary Ion Mass Spectrometry (SIMS) method, a Fast Atom Bombardment (FAB) method, a matrix-assisted laser desorption ionization (MALDI) method, an electrospray ionization (ESI) method, or the like can be used. The analysis unit may use a double-focus mass spectrometer, a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a fourier transform mass spectrometer, an ion cyclotron mass spectrometer, or the like. For precise analysis, a tandem mass spectrometer (MS/MS) in which two mass spectrometers are combined may be used.

The mass spectrometer may be used alone, or may be connected to a separation device such as a liquid chromatography, a measurement device, or the like, and can perform analysis by a liquid chromatography-mass spectrometry (LC/MS ) combination with high performance liquid chromatography. Mass spectrometry can be performed by Selective Reaction Monitoring (SRM) or Multiplex Reaction Monitoring (MRM) using a triple quadrupole mass spectrometer, which is an LC/MS (LC/MS) system widely used in the art for the quantitative detection of peptides. By SRM/MRM, multiple factors can be measured simultaneously, and hundreds of proteins and peptides can be measured simultaneously by a single measurement.

When the presence of the biomarker protein or the partial peptide in a large amount in a sample collected from a subject is positive, the subject can be determined to have colorectal cancer.

In the present invention, a sample collected from a healthy normal person may be simultaneously measured as a negative object. In this case, since the concentration of the biomarker protein or the partial peptide in the sample of the subject is higher than that in a healthy normal person when the subject has colorectal cancer, the subject can be determined as having colorectal cancer by determining that the biomarker protein or the partial peptide is positive when the concentration of the biomarker protein or the partial peptide in the sample of the subject is higher than that in a healthy normal person. Whether or not a biomarker protein or a partial peptide in a sample is positive can be determined by determining a cut-off value in advance for the ratio of the area value of the peak of the biomarker peptide detected by the SRM/MRM method to the area value of the peak of the stable isotope-labeled peptide as an internal standard or for the antibody measurement value, and determining that the sample is positive when the cut-off value is exceeded based on the cut-off value.

The cut-off value is determined, for example, by ROC (receiver operating characteristic curve) analysis. In addition, the diagnostic accuracy (sensitivity and specificity) by the method of the present invention can be determined by ROC analysis. ROC analysis measures a biomarker protein or a partial peptide in a sample collected from a colorectal cancer patient as a sample and a sample collected from a healthy normal person, calculates sensitivity and a false positive rate (1-specificity) at each cutoff value, and plots the calculated sensitivity and false positive rate on a coordinate where the horizontal axis represents the 1-specificity and the vertical axis represents the sensitivity.

When the results of measurement by the method of the present invention are analyzed for diagnostic accuracy by ROC analysis, the area under the curve (AUC) is as high as 0.9 or more, the sensitivity is 70% or more, preferably 80% or more, more preferably 85% or more, further preferably 90% or more, and the specificity is 70% or more, preferably 75% or more, further preferably 80% or more. The method of the present invention can detect early colorectal cancer with very high accuracy.