JP5570810B2 - Colorectal cancer marker polypeptide and diagnostic method for colorectal cancer - Google Patents

Description

  The present invention relates to a colorectal cancer marker polypeptide, particularly a colorectal cancer marker polypeptide present in serum or the like, and a method for diagnosing colorectal cancer by detecting the colorectal cancer marker polypeptide.

  Along with lung cancer, stomach cancer, and liver cancer, colorectal cancer has a high incidence, and the incidence is rapidly increasing with the recent westernization of dietary habits. Conventionally, colorectal cancer has been subjected to fecal occult blood test, X-ray contrast test, colonoscopy, etc., followed by diagnosis by biopsy histological examination.

  However, the fecal occult blood test is intermittent because bleeding from colorectal cancer is intermittent, so the rate of false negative judgments is high. Conversely, hemorrhoids, gingivitis, ulcers, and aspirin use cause positive judgments. Therefore, there is a drawback that unnecessary invasive reexamination is performed. In addition, X-ray contrast examinations and colonoscopy are expensive, and have the disadvantage of burdening and distressing patients.

  Therefore, research was conducted on a method for examining colorectal cancer that is non-invasive and does not cause pain to the patient. For example, there is a method (Patent Document 5) for detecting the presence of advanced colorectal cancer by detecting carcinoembryonic antigen (CEA) from a blood sample. However, in a reliable test for detecting an early colorectal tumor, Absent. In addition, a method for diagnosing cancer including colorectal cancer (Patent Document 1) for identifying a certain colorectal cancer-related polypeptide as an antigen showing an immune response in colorectal cancer, colorectal cancer using cDNA encoding a colorectal cancer marker And a method for diagnosing colon diseases such as polyps (Patent Document 2), a method for detecting colorectal cancer using a colon cancer marker gene encoding a protein having a specific glycosyltransferase activity (Patent Document 3), and an APC gene, Non-invasive early detection of colorectal cancer cells and / or colorectal cancer progenitor cells using primers that can analyze mutations in selected regions of the K-ras gene, β-catenin gene and B-raf gene (Patent Document 4) and the like. However, these colorectal cancer test methods have not changed clinical diagnosis methods in terms of accuracy, ease of implementation, and cost.

Special table 2004-534218 Special Publication 2004-537971 Publication JP 2006-136319 A Special Table 2007-505609 U.S. Pat.No. 005,741,650 A

  The present invention relates to a colorectal cancer marker polypeptide, particularly a colorectal cancer marker polypeptide present in serum or the like, a colorectal cancer diagnostic method for detecting the colorectal cancer marker polypeptide, an antibody for the diagnosis, and a kit used for the diagnostic method The purpose is to provide.

  As a result of investigations to obtain a colorectal cancer marker useful for diagnosis, the present inventors have found that it is inefficient to perform a test on a cellular specimen, particularly a cell obtained by biopsy, and imposes a burden on the subject. Therefore, research was conducted on the assumption that a colon cancer marker contained in a body fluid sample, particularly serum or the like is preferably used for early colon cancer diagnosis and can be easily examined. As a result, the inventors have found that the serum of colorectal cancer patients contains four types of peptides serving as colorectal cancer markers.

  Therefore, the present invention provides a colorectal cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5. In the present specification, a colorectal cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, or 5. They are referred to as colon cancer marker polypeptides A, B, D, and C, or colon cancer markers A, B, D, and C, respectively.

The present invention also includes the following polypeptide (1) or (2) that induces antibody production against a colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5. Polypeptide fragments are provided.
(1) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5; and
(2) A polypeptide comprising an amino acid sequence mutated from the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5 by substitution, deletion, addition and / or insertion of one or more amino acid residues.

Furthermore, the present invention detects at least one colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5 from a body fluid sample obtained from a subject, particularly serum or plasma. A diagnostic method for colorectal cancer is provided.
Furthermore, the present invention detects at least one colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5 from a body fluid sample obtained from a subject, particularly serum or plasma. A diagnostic method for colorectal cancer metastasis is provided.

Furthermore, the present invention provides an antibody that recognizes a colon cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5.
Furthermore, the present invention provides an antibody that recognizes a colon cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5, comprising administering the polypeptide fragment to a non-human mammal. A method of manufacturing the same is provided.

Furthermore, the present invention provides a kit for diagnosing colorectal cancer, comprising at least one antibody that recognizes a colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5. .
Furthermore, the present invention is for diagnosing colorectal cancer metastasis, comprising at least one antibody having specificity for a colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5. Provide kit.

(Definition)
In the present specification, “low molecular weight protein” refers to a protein having a molecular weight of 30,000 or less, preferably 20,000 or less. Further, unless otherwise specified, “low molecular weight protein and the like” means both low molecular weight proteins and peptides. Examples include physiologically active proteins such as peptide hormones, interleukins, and cytokines that are present in trace amounts, and trace amounts of biomarker proteins and peptides that have no particular function. Since they pass through the kidney and are partially excreted in the urine, it may be possible to measure not only blood but also urine as a sample.

In the present specification, “plasma” refers to a supernatant in which EDTA or heparin is added and centrifuged to which a blood coagulation system hardly acts or does not act.
In the present specification, “serum” is a portion obtained by removing a coagulation component from blood. If fresh blood is allowed to stand, blood coagulation occurs, and then blood cells and fibrin contract in a lump and release a clear amber serum. Its component is almost equal to the plasma minus fibrinogen.

In the present specification, “major protein” such as serum and “carrier protein” refer to a high content protein in serum. Examples include albumin (molecular weight 66 kDa), immunoglobulin (150-190 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several hundred kDa) and the like.
In this specification, “SDS” means sodium dodecyl sμlfate.
In the present specification, “PAGE” means polyacrylamide gel electrophoresis.

(Search for colorectal cancer marker polypeptide)
In order to discover colorectal cancer markers that are useful for early detection of colorectal cancer and that are not collected in cell samples such as biopsy, but are present in easily collected body fluid samples, the present inventors have collected serum collected from colorectal cancer patients. The colorectal cancer marker was searched using it. The method for searching, identifying and evaluating the colorectal cancer marker is as described in the examples described later.
The final identification of the colon cancer marker polypeptides A, B, D and C of the present invention was performed by tandem MS (MS / MS) mass spectrometry as shown in Example 4. Next, the concept of the mass spectrometry will be described.

  FIG. 1 shows the cleavage pattern of a 4-residue peptide. The name of the cleaved peptide varies depending on the cleavage site. For example, a peptide fragment cleaved at the peptide-binding portion of the main chain is B1 for a peptide fragment containing 1 amino acid residue at the N-terminus of the peptide, B2 for a peptide fragment containing 2 amino acid residues including the N-terminus, and similarly from the N-terminus A substance containing 3 amino acid residues is called B3. On the other hand, one amino acid residue at the C terminal of the peptide is called Y1, a peptide fragment containing two amino acid residues including the C terminal is called Y2, and a peptide fragment containing three amino acid residues is called Y3. The peptide fragments B1 to B3 are called B series, and the peptide fragments Y1 to Y3 are called Y series. Similarly, peptide fragments cleaved by A, X, C, and Z are also called A series, X series, C series, and Z series, and are numbered according to the number of amino acid residues contained.

The MS / MS of the MALDI-TOF-MS analyzer used this time is a low energy dissociation, and the B series and Y series appear strongly because it is mainly cleaved at the peptide bond. In the MS / MS spectrum of FIG. 1, signals (output data) corresponding to m / z of the peptide fragments of the B series and the Y series are strong. Since the A, X, C, and Z sequences increase in the cleavage with a larger cleavage energy, the MS / MS spectrum becomes more complicated. The energy of cleavage depends on the type of mass spectrometer. UCSF provides a mass spectrum analysis homepage (ProteinProspector: http://prospector.ucsf.edu/).
Mass, MS / MS patterns, and protein enzyme digestion patterns can be calculated.
The colorectal cancer marker polypeptide of the present invention was identified by analyzing output data corresponding to m / z of the polypeptide and m / z of the B-series and Y-series peptide fragments, and corresponds to Example 4. FIG. 13, FIG. 14 and FIG. 15 show the output data list.

(Detection of colorectal cancer marker polypeptide)
The colorectal cancer marker of the present invention is a colorectal cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5, and this is detected from a body fluid sample of a subject to be examined, particularly serum or plasma. By doing so, diagnosis of colorectal cancer and / or metastasis can be determined.
The detection method used in the present invention is not particularly limited. For example, an antibody against the colorectal cancer marker polypeptide is prepared, and a method for detecting the colorectal cancer marker of the present invention by an antigen-antibody reaction with the antibody, or recent progress has been made. Good proteomic approach.

(Intestinal cancer polypeptide marker detection antibody)
The antibody of the present invention is a polyclonal antibody or a monoclonal antibody that can be used for detection of a colon cancer marker polypeptide. In particular, monoclonal antibodies can be selected with excellent specificity.
Immunization can be performed by a conventional method. For example, it is preferable to administer an antigen 2 to 3 times at intervals of 7 to 30 days, for example. The dose of the antigen is about 0.05 to 2 mg per mouse, for example. The administration route is not particularly limited, and can be appropriately selected from subcutaneous, intradermal, intraperitoneal cavity, vein, and intramuscular. The antigen can be used after being dissolved in an appropriate buffer, for example, a complete buffer containing a commonly used adjuvant such as Freund's complete adjuvant or aluminum hydroxide.

  After the immunized mammal has been bred for a certain period of time, it can be boosted with 100 μg to 1000 μg of antigen after the antibody titer has risen. Blood is collected from a mammal immunized usually 1 to 2 months after the last administration, and the blood is subjected to, for example, centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, A polyclonal antibody that recognizes the colorectal cancer marker polypeptide of the present invention can be obtained as a polyclonal antiserum by separation and purification by a conventional method such as chromatography such as affinity chromatography.

  Monoclonal antibodies can be obtained by cloning antibody-producing cells. In general, antibody-producing cells recovered from immunized animals are cell-fused with an appropriate fusion partner to form a hybridoma. After selecting a hybridoma of a cancer cell using a HAT culture solution, the activity of the antibody produced is used as an indicator. As a screening. Subsequently, the obtained hybridoma can be cultured in an appropriate medium according to a conventional method, and then the culture solution can be purified to obtain a monoclonal antibody.

  Examples of immunized animals that can be used in the present invention include non-human mammals such as monkeys, sheep, goats, pigs, dogs, rats, mice, rabbits, and particularly rodents such as rats, mice, and hamsters. preferable. When mice are used as immunized animals, mouse-derived myeloma cells are preferred as fusion partners.

As an antigen used to sensitize the non-human mammal, it induces antibody production against a colon cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5, and the following (1 ) Or a polypeptide fragment containing the polypeptide of (2) can be used.
(1) a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5; and
(2) A polypeptide comprising an amino acid sequence mutated from the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5 by substitution, deletion, addition and / or insertion of one or more amino acid residues.

In addition, the antigen may be the polypeptide fragment containing at least 15 amino acid residues, preferably at least 20 amino acid residues, and more preferably at least 25 amino acid residues.
These polypeptide fragments can be prepared by introducing a vector incorporating a cDNA encoding the fragment into a host, or by chemical synthesis.

(Making by recombination method)
In the recombination method, for example, a vector incorporating the cDNAs of SEQ ID NOS: 7 to 14 described in the present specification is prepared, introduced into a suitable host cell, transformed, the transformed cell is cultured, and a culture solution or the like is obtained. By purifying, a polypeptide fragment used for inducing the antibody can be prepared.

The host cell is not particularly limited as long as it can express a mammalian gene, and cells such as vertebrates, insects and yeasts can be used. For example, in the case of vertebrate cells, COS cells that are monkey cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCC CRL-1650), Chinese hamster ovary cells (CHO cells, ATCC CCL-61) ) And its dihydrofolate reductase deficient strain (Urlaub,
G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220), mesenchymal stem cells and osteochondral cultured cell lines can be used. For insect cells, Spodoptera frugiperda ovarian cell-derived cell lines (Sf-9 or Sf-21) and Trichoplusia ni egg cell high five cells (Wickham, TJ et al, (1992) Biotechnol Prog.i: 391-396) can be used.

  As an expression vector for vertebrate cells, a vector having a promoter located upstream of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. Examples of the expression vector include PCR3.1 having a cytomegalovirus early promoter (Invitrogen), pSV2dhfr having an SV40 early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) and the like, retroviral vector pLCNX (Clontech, K1060-1) and the like can be used.

  As an example of the case where a COS cell is used as the host cell, the expression vector has an SV40 origin of replication, can be independently propagated in the COS cell, and further comprises a transcription promoter, a transcription termination signal, and an RNA splice. The thing provided with the site | part is preferable. The expression vector includes diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and van der Eb, AJ (1973) Virology 52, 456-457) and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845) and the like can be incorporated into COS cells.

  When CHO cells or 293 cells are used as host cells, a vector capable of expressing a neo gene that functions as an antibiotic G418 resistance marker together with an expression vector, such as pRSVneo (Sambrook, J. et al. (1989) : "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory, NY) and pSV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) By selecting G418-resistant colonies, transformed cells that stably produce the polypeptide of interest can be obtained.

In addition, when insect cells are used as host cells, cell lines derived from ovarian cells of Spodoptera frugiperda (Sf-9 or Sf-21) of Lepidoptera gourdaceae and high five cells derived from egg cells of Trichoplusia ni (Wickham, TJ et al , (1992) Biotechnol. Prog.i: 391-396) is often used as a host cell, and the baculovirus transfer vector is pVL1392 / 1393 which uses the promoter of the polyhedrin protein of autographer nuclear polyhedrosis virus (AcNPV). (Kidd, i. M. and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 420, 137-159). Furthermore, vectors using baculovirus P10 and promoters of the same basic protein can also be used. Furthermore, it is also possible to express a recombinant protein as a secreted protein by linking the secretory signal sequence of AcNPV envelope surface protein GP67 to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998)). Biol. Chem., 379, 167-174).
The recombinant cells produce the desired protein on their cell membranes by culturing according to a conventional method. As a medium used for the culture, various media commonly used according to the employed host cells can be appropriately selected.

(Created by chemical synthesis method)
Chemical synthesis of the polypeptide fragment can be performed by chemical methods well known in the art. For example, peptide synthesis using solid phase technology can be performed by a batch or continuous flow process. In a continuous flow process, alpha amino protected and side chain protected amino acid residues are continuously added to an insoluble polymer support via a linker. A linker, such as methylamine derivatized polyethylene glycol, is attached to poly (styrene-co-divinylbenzene) to form a support resin. This amino acid residue is N-α- protected by the acid labile Boc (t-butyloxycarbonyl) method or the base labile Fmoc (9-fluorenylmethoxycarbonyl) method. The carboxyl group of the protected amino acid is attached to the linker amine, and this residue is attached to the solid support resin. When Boc or Fmoc is used, the protecting group is removed using trifluoroacetic acid or piperidine. Each additional amino acid is added to the bound residue using a coupling reagent or a pre-activated amino acid derivative, and then the resin is washed. The full-length peptide is synthesized by stopping successive protections, ie derivatized amino acids, and washed with dichloromethane and / or N, N-dimethylformamide. This peptide is cleaved between the peptide carboxyl terminus and the linker to produce a peptide acid or peptide amide (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego CA pp. S1-S20). Peptides can be synthesized automatically using equipment such as the ABI 431 A peptide synthesizer (Applied Biosystems). The synthesized polypeptide fragment can be purified by high performance liquid chromatography and its composition can be confirmed by amino acid analysis or sequencing (Creighton (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY).

  The obtained antibody is preferably assayed using ELISA or liposome lysis assay. In ELISA, a microplate sensitized with an antigen whose reactivity is to be observed is prepared, and a sample obtained by appropriately diluting the culture supernatant of the hybridoma is added thereto and reacted. After sufficiently reacting, the well is washed and further reacted with a secondary antibody against immunoglobulin. When the secondary antibody finally bound to the well is measured, the binding activity of the antibody present in the culture supernatant to the antigen can be quantitatively known.

  The liposome lysis assay is a method that utilizes the phenomenon that when an antibody reacts with an antigen-sensitized liposome, the liposome is dissolved by the action of complement. The liposome is composed of, for example, dicetyl phosphate (hereinafter abbreviated as DCP), dimyristoyl phosphatidylcholine (hereinafter abbreviated as DMPC), cholesterol, and a phospholipid antigen to be tested. When these lipid components are dissolved in a suitable organic solvent and then dried to form a lipid film, and added to the aqueous solvent and stirred vigorously, multilayered lamellar liposomes are formed. In the liposome thus prepared, a phospholipid antigen is incorporated as a component of the membrane. Therefore, it can be expected that the antigen structure is presented in a state close to the shape present in the actual cell membrane, and thus it is suitable for screening for monoclonal antibodies. It is preferable to encapsulate the inside of the liposome with a fluorescent dye that serves as a label for dissolution. As the fluorescent dye, 4-methylumbelliferone, calcein, or the like is used. When an antibody binds to the phospholipid antigen constituting the liposome in the presence of complement, the liposome is destroyed and the fluorescent dye inside flows out, so that the presence of the antibody is observed as an increase in fluorescence in the liquid phase.

(Detection of colorectal cancer marker polypeptide)
In the present invention, at least one colorectal cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5 is detected from a body fluid sample obtained from a subject, preferably serum or plasma. Provides a method for diagnosing colorectal cancer. When the marker of this invention exists in the quantity which shows 1 or more types positive, it can be considered that it is suffering from colon cancer or it has reached the previous stage.

  Also, a body fluid sample obtained from a subject from which at least one colorectal cancer marker polypeptide comprising the amino acid sequence of SEQ ID NOs: 1, 2, 3, and 5 has been removed surgically or by other means is removed. By detecting from this, metastasis of colorectal cancer can be diagnosed. That is, if the colon cancer is removed, the serum of the subject usually does not have the colon cancer marker, or the abundance is low, but if the colon cancer has metastasized to other than the removed site by metastasis, the colon cancer The amount of cancer marker is positive.

(Detection method using specific antibody)
The colon cancer marker polypeptide of the present invention can be detected by contacting a polyclonal antibody or a monoclonal antibody obtained by the method described above with a body fluid sample obtained from a subject. The antigen-antibody reaction can be performed by a known ordinary method. Examples of such conventional methods include ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), and fluorescence activated cell sorter method (FACS). The detection of the colon cancer marker polypeptide is preferably a two-site monoclonal immunoassay using monoclonal antibodies that react with two non-interfering epitopes, but a competitive binding assay can also be used (Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New
York NY).

  In order to carry out the detection method using the antibody, the present invention contains at least one antibody that recognizes a colorectal cancer marker polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5. A colorectal cancer diagnostic kit and a colorectal cancer metastasis diagnostic kit are provided.

(Detection method using proteomic method)
Since the colorectal cancer marker polypeptides A, B, D, and C of the present invention are known for their amino acid sequences and molecular weights, they can be detected from a body fluid sample such as serum obtained from a subject by mass spectrometry. The mass spectrometry can be performed by liquid chromatography mass spectrometry (LC-MS analysis) or matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS analysis).

  When detection is performed by MALDI-TOF-MS analysis, mass spectrometry data of proteins and polypeptides contained in a liquid sample is obtained, and the output corresponding to the mass corresponding to the colorectal cancer marker of the present invention (peak) ), The presence or absence of a colorectal cancer marker can be examined.

  The procedure for examining the presence or absence of colorectal cancer markers by the MALDI-TOF-MS analysis is as follows. First, a body fluid sample is pretreated, and the protein and polypeptide components contained in the sample are concentrated or lyophilized, and then mixed with an appropriate matrix and dried on a sample plate. The sample is introduced into the high vacuum of the mass spectrometer. The sample spot is irradiated with a laser to desorb ions into the gas phase, and a time measuring device for measuring the time of flight is started. Ions are accelerated by the electric field with the same kinetic energy and are separated corresponding to the mass-to-charge ratio while flying through the free flight tube in the electric field. The ions reach the detector at different times depending on the mass to charge ratio of the ions. By controlling all the parameters of the detector by the data system and acquiring the detection signal for each time, it is possible to process data indicating the arrival order of ions depending on the mass-to-charge ratio. After obtaining a profile of the protein and polypeptide contained in the sample with a predetermined spectrometer, the colon cancer marker is detected by examining the presence of output data corresponding to the colorectal cancer marker polypeptide of the present invention in the profile. can do.

A comparative colorectal cancer marker polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5 labeled with an isotope element such as an oxygen atom, a carbon atom, a hydrogen atom and a nitrogen atom is used. This improves the accuracy of detection (qualitative) and enables quantitative analysis.
For example, as shown in FIG. 17, the standard marker polypeptide A for comparison is obtained by substituting the carbon atoms of alanine and glycine of the colon cancer marker polypeptide A of the present invention with ¹³ C carbon atoms, which are isotopes. be able to. Then, by adding a certain amount of the standard polypeptide marker A to the sample and performing mass spectrometry, reference output data of intensity 1 can be obtained as shown in FIG. Since the reference output data always appears at a certain position, it becomes a reference for colorectal cancer marker polypeptide A detection (qualitative). Further, by comparing with the reference output data as shown in FIG. 18, it is possible to clearly confirm the decrease in the colorectal cancer marker polypeptide A after the colorectal cancer excision surgery. Further, if a decrease in colorectal cancer marker polypeptide A is not confirmed after colorectal cancer removal surgery, metastasis of the colorectal cancer is suspected.

(Pretreatment of body fluid samples, especially serum and plasma)
Prior to detecting the colorectal cancer marker polypeptide, it is preferable to remove the major protein from the body fluid sample, particularly serum and plasma, and concentrate the colorectal cancer marker polypeptide. Serum obtained from a subject contains a major protein having a relatively high molecular weight, which may interfere with detection of the colorectal cancer marker polypeptide of the present invention or significantly reduce detection sensitivity. In particular, when a proteomic method is used for the detection, the influence of main proteins including a carrier protein is great. For the removal of the main protein, the method described in Japanese Patent Application No. 2007-206602 filed on August 8, 2007 by the present inventors is preferable. That is,
(a) Reagent 1 containing urea and thiourea and reagent 2 containing dithiothreitol (DTT) are added to and mixed with a body fluid sample obtained from a subject, particularly serum, etc., and then reagent 3 containing 90% or more of an organic solvent Dropping and mixing with
(b) a step of stirring the mixed solution obtained in the step (a) at a low temperature;
(c) centrifuging the stirred solution obtained in step (b) at low temperature and removing the supernatant;
(d) adding the reagent 4 containing an organic solvent and an acid to the precipitate obtained in (c) and mixing;
(e) stirring the mixed solution obtained in step (d) at a low temperature; and
(f) The stirring solution obtained in the step (e) is centrifuged at a low temperature, and the main protein including the carrier protein is removed by processing in a step of collecting the supernatant. The method may further include a step (g) of lyophilizing the supernatant collected in step (f).

  The concentration in reagent 1 is urea 1-8M, and thiourea 0.5-3M, preferably urea 3-8M, and thiourea 1-3M, and the concentration of DTT in reagent 2 is 5 mM-10M, preferably Is 20 mM to 1 M. The organic solvent of the reagent 3 is selected from the group consisting of acetone, ethanol, methanol, 2 propanol, acetonitrile, and a mixture thereof, preferably acetone.

  The organic solvent of reagent 4 is selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol and mixtures thereof, preferably acetonitrile, and the acid is hydrochloric acid, trifluoroacetic acid (TFA), formic acid. , Acetic acid, and trichloroacetic acid (TCA), and the concentration of the organic solvent is 50 to 99%. A preferable acid of reagent 4 is hydrochloric acid, and its concentration is preferably 5 mM to 500 mM.

  Further, the lyophilized product of the supernatant obtained in the step (g) contains a component selected from the group consisting of TFA, hydrochloric acid, formic acid, acetic acid, and TCA, preferably 0.1 to 20% of TFA. The reagent 5 to be analyzed can be added to prepare a sample for analysis of the colorectal cancer marker polypeptide of the present invention.

  Eight healthy subject sera and eight colorectal cancer-affected sera were prepared, and colon cancer marker polypeptides were searched according to the flowchart shown in FIG. Subsequently, the obtained colorectal cancer marker polypeptide was evaluated.

Example 1 Concentration of Colorectal Cancer Marker Polypeptide Component In this example, the colorectal cancer marker polypeptide component was concentrated according to the flowchart shown in FIG. 3 (hereinafter, this concentration method is referred to as “K method”).
First, 10 μl of the prepared serum sample was collected, 18 μl of reagent 1 (7M urea, 2M thiourea) and 2 μl of reagent 2 (200 mM DTT) were added, and mixed with Vortex. At that time, the serum, Reagent 1 and Reagent 2 were all cooled to 4 ° C. and used. Subsequently, the mixed solution was dropped into 1.2 ml of reagent 3 composed of high-purity acetone cooled to 4 ° C., and immediately after the dropping, the mixture was mixed using a vortex, and then stirred at 4 ° C. for 1 hour. After the stirring, the mixed solution was centrifuged at 19 ° C. for 15 minutes with a cooling centrifuge (19,000 g), and the resulting supernatant was completely removed. Next, 200 μl of 4 ° C. reagent 4 (acetonitrile 70%, hydrochloric acid 12 mM, balance water) was added to the remaining precipitate, and the mixture was stirred at 4 ° C. for 1 hour, and then the mixed solution was centrifuged at 4 ° C. for 15 minutes with a cooling centrifuge. After separation (19,000 g), the resulting supernatant was recovered as a serum polypeptide concentrated solution.
Subsequently, the concentrated solution was freeze-dried, and 80 μl of reagent 5 (99.9% H ₂ O, 0.1% TFA) was added to the resulting freeze-dried product to dissolve it.

(Example 2) Fractionation of serum polypeptide by reversed-phase HPLC Polypeptide-concentrated solutions of healthy subject serum (N1-N8) and colorectal cancer patient serum (P1-P8) obtained in Example 1 were each reversed-phase. Fractionated by HPLC. The fraction was fractionated into 60 fractions at intervals of 1 minute, with an elution time of 25 to 85 minutes of reverse phase HPLC. The analysis conditions are shown in FIG. 4, and the analysis results are shown in FIG.

Example 3 Search for Polypeptides of Fractions Obtained by Reversed Phase HPLC Each fraction obtained in Example 2 was subjected to MALDI-TOF-MS (Matrix-Assisted Laser Desorption Ionization Time-Of-Fright Mass Spectrometer:
Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer, model number: Voyager-DE PRO, manufacturer: Applied Biosystems, USA) and search for polypeptides that are specifically present in sera P1 to P8 of colorectal cancer patients did. First, 1 μl of each fraction sample and 0.5 μl of matrix were mixed and air-dried on a MALDI-TOF-MS sample plate, and then MALDI-TOF-MS analysis was performed. As the matrix, α-cyano-4-hydroxycinnamic acid (CHCA; manufactured by Nacalai Tesque) was used.
A saturated solution dissolved in 50% CH ₃ CN-0.1% TFA was used.

  The presence of polypeptides of healthy subject serum N1 to N8 and colon cancer patient sera P1 to P8 in each fraction, as shown by the MALDI-TOF-MS analysis, are shown in FIGS. White circles in each figure indicate polypeptides present in healthy subject sera N1 to N8, and gray circles indicate polypeptides present in colon cancer patient sera P1 to P8. Each circle is displayed by sliding it little by little to clarify its existence, and the size of each circle indicates the intensity of the peak of MALDI-TOF-MS analysis. Moreover, the vertical axis | shaft of FIGS. 6-9 shows a mass / valence, and a horizontal axis shows the number of the fraction of HPLC.

  As a result of examining the polypeptide distribution of each fraction shown in FIG. 6 to FIG. 9, it was not found in healthy subjects in the sixth fraction, the fifteenth fraction, and the eighteenth fraction, and was specific to colon cancer patients. Polypeptide distribution was seen. That is, FIG. 10 shows MS spectra of four types of peptides increasing specifically in colorectal cancer patient serum. The spectrum in the figure is as follows.

  As shown in FIG. 10, according to the spectrum of the MALDI-TOF-MS method, a part of the spectrum of fraction No. 6 (m / z = 2092, and 2163) of each sample, fraction No. 15 Serospecific peptides of colorectal cancer patients are detected in part of the spectrum (m / z = 2188) and part of the spectrum of fraction No. 18 (m / z = 3505).

  The analysis results of the specific peptide are shown in FIGS. FIG. 11 is an average spectrum of the MS spectra (serious human serum N1 to N8, colon cancer patient sera P1 to P8) of each colon cancer marker polypeptide of FIG. FIG. 12 shows a graph (left) representing the mean and standard deviation of the intensity of these MS peaks and the P value (right) obtained from the intensity of the MS peak of each colorectal cancer marker polypeptide. For / z = 2092 (A), m / z = 2163 (B), m / z = 2188 (C), and m / z = 3505 (D), they were 0.0013, 0.0021, 0.0006, and 0.0047, respectively. The polypeptide corresponding to these results was determined to be a colon cancer marker polypeptide.

(Example 4) Identification of colorectal cancer marker polypeptide The colorectal cancer marker polypeptides A, B, D and C obtained in Example 3 were identified.
(Concentration and fractionation of colorectal cancer patient serum)
In the identification, in order to increase the intensity of the MS spectrum, the polypeptide and the like were concentrated by the same method as in Example 1 except that 20 μl of colorectal cancer patient serum was used.

  That is, according to the flow chart of the K method shown in FIG. 3, 20 μl of colorectal cancer serum sample was collected, 36 μl of reagent 1 (7M urea, 2M thiourea) and 4 μl of reagent 2 (200 mM DTT) were added and mixed with Vortex. At that time, the serum, Reagent 1 and Reagent 2 were all cooled to 4 ° C. and used. Subsequently, the mixed solution was dropped into 1.8 ml of reagent 3 made of high-purity acetone cooled to 4 ° C., and immediately after the dropping, the mixture was mixed using vortex, and then stirred at 4 ° C. for 1 hour. After the stirring, the mixed solution was centrifuged at 19 ° C. for 15 minutes with a cooling centrifuge (19,000 g), and the resulting supernatant was completely removed. Next, add 800 μl of reagent 4 (acetonitrile 70%, hydrochloric acid 12 mM, balance water) at 4 ° C. to the residual precipitate, stir at 4 ° C. for 1 hour, and then centrifuge the mixed solution at 4 ° C. for 15 minutes with a cooling centrifuge. After separation (19,000 g), the resulting supernatant was recovered as a serum polypeptide concentrated solution.

Subsequently, the concentrated solution was freeze-dried, and 80 μl of reagent 5 (99.9% H ₂ O, 0.1% TFA) was added to the resulting freeze-dried product to dissolve it.
Subsequently, the obtained serum polypeptide concentrated solution was fractionated by reverse phase HPLC. The fraction was fractionated into 60 fractions at intervals of 1 minute, with an elution time of 25 to 85 minutes of reverse phase HPLC. The analysis conditions are as shown in FIG.

(MS and MS / MS spectrum measurement of polypeptide markers)
10 μl of lyophilized fractions (A, B: fraction No. 6, C: fraction No. 15, and D: fraction No. 18) in which the polypeptide markers A, B, C and D are present In a solvent (50% acetonitrile / 0.1% TFA). 1 μl of the solution was taken and subjected to MALDI-TOF-MS analysis, and MS spectra and MS / MS spectra of the peptide markers A, B, C and D were measured. The measurement was performed using MALDI-TOF-MS (Autoflex II TOF / TOF: Bruker Daltonics, USA) capable of measuring a tandem MS spectrum (MS / MS spectrum).

(MS and MS / MS spectrum analysis)
From the MS spectra (exact molecular weight) and MS / MS spectra of the peptide markers A, B, C and D obtained by the measurement, using MS spectrum analysis software Mascot (Matrix Science, USA), NCBI database Each peptide marker was identified by searching with. The Mascot Scores of peptide markers A, B, D, and C are 126, 106, 92, and 162, respectively. From this result, it is considered that the reliability of identification of each peptide is extremely high.

In addition, the identification procedure of each peptide marker using the MS spectrum analysis software Mascot of this example was performed according to the following procedure.
First, from the amino acid sequences of all proteins in the NCBI database, all peptide fragments that match the molecular weight obtained from the MS spectrum of the target peptide marker within a certain error range are listed. Here, the error range depends on the mass accuracy of the MS spectrum measurement. In this example, the observed error range was m / z ± 100 ppm (m / z corresponds to the ratio of mass to ion valence, and the horizontal axis of the MS spectrum corresponds to the value of).

  The MS / MS spectra of all the listed polypeptides were then simulated. Here, the simulated MS / MS spectrum and the measured MS / MS spectrum of the target polypeptide were compared, and the degree of coincidence was evaluated as a score. The listed polypeptides are displayed with a score from the highest score to the protein containing the peptide and its amino acid sequence.

  The identification results of each polypeptide are shown in FIGS. 13, 14 and 15, respectively. From these results, it was revealed that the colon cancer peptide markers A, C, and D are polypeptides corresponding to SEQ ID NOs: 1, 5, and 3. 13, 14 and 15 show output data lists corresponding to B / Y series m / z of peptide fragments corresponding to colon cancer marker polypeptides A, C and D. The colorectal cancer marker of the present invention is identified by analyzing the output data. Although there is no data on colorectal cancer marker polypeptide B, polypeptide B has an Ala residue on the N-terminal side of polypeptide A, and is identified by “Mascot Search and polypeptide A MS / MS. The amino acid sequence could be confirmed from the coincidence of the B-series fragments in the spectrum.

  Furthermore, as a result of preparing chemically synthesized products of colorectal cancer polypeptide markers A, C and D and performing MS and MS / MS analysis, the same MS / as the colorectal cancer polypeptide markers A, C and D obtained from the serum. MS spectrum was observed. From this, it was confirmed that the identification results of the colon cancer marker polypeptides A, C and D were correct. These MS / MS spectral data are shown in FIG.

(Example 5) Evaluation of colorectal cancer peptide marker
The colorectal cancer peptide marker of the present invention was evaluated by quantitative analysis of the abundance of peptide marker A in the serum of colorectal cancer patients before and after colorectal cancer excision surgery.
Using a stable isotope ( ¹³ C) -labeled peptide marker A, quantitative analysis was performed on the abundance of peptide marker A in the serum before and after surgery to remove colorectal cancer in one colorectal cancer patient. The experimental flowchart is shown in FIG. In this method, the carbonyl carbon in the main chain of some amino acids in the polypeptide was substituted with a stable isotope carbon ( ¹³ C). For this reason, properties other than mass, that is, concentration efficiency by the K method, elution time of reverse phase HPLC, and ionization efficiency during mass analysis are the same. Therefore, as shown in FIG. 16, by introducing a certain amount of ¹³ C-labeled peptide A into the serum before the K method treatment and comparing the intensities of peptide marker A and ¹³ C-labeled peptide marker A on the MS spectrum, The amount of peptide marker A present in the original serum can be quantified.

FIG. 18 shows the result of quantitative analysis of the concentration of peptide marker A contained in the serum of the same patient before and after colorectal cancer excision surgery using ¹³ C-labeled peptide marker A. The intensity ratio of the MS spectrum of ¹³ C-labeled peptide marker A and peptide marker A originally present in serum was 0.63: 1 before surgery, but decreased to 0.15: 1 after surgery. This shows that the concentration of peptide marker A present in the serum was 15.8 fmol / ml before the operation, but decreased to 3.8 fmol / ml after the operation. As a result of repeating this experiment four times, the reproducibility was very high and the CV value was 5% or less.
From this result, the abundance of peptide marker A in serum correlates with the presence or absence (or abundance) of colorectal cancer in the body, and can be evaluated as having sufficient effectiveness as a colorectal cancer diagnostic marker.

該大腸癌患者の血清に、該血清１μｌ当たり、安定同位体標識ペプチドA2，C2及びD2をそれぞれ５０fmol、２．５fmol及び２５fmolを加えた。次いで、実施例１と同じ方法で前記大腸癌マーカポリペプチド成分を濃縮し、実施例２と同じ方法で該ポリペプチド成分を逆相HPLCにより６０分画に分取した。ポリペプチドマーカＡ，Ｃ及びＤを含む分画を濃縮した後、MALDI-TOF-MS分析を行った。該分析データを図22に示した。データＡから、ポリペプチドマーカＡの血清中濃度は約120 ng/ml、データＣからポリペプチドマーカＣは約４ng/ml、かつデータＤからポリペプチドマーカＤは約60 ng/mlであることが判った。(Example 6) Quantification of three types of polypeptide markers A, C and D in the serum of one colorectal cancer patient and the results
First, peptides A, C and D of the present invention and their stable isotope labeled peptides were prepared. The labeled peptide and isotopes used for labeling are as follows.
(Stable isotope labeled peptide)
* Chemically synthesized stable isotope-labeled peptide A using the amino acid underlined as the stable isotope-labeled amino acid (FMOC form) in the table below
DE AG SE A DHE G THSTKR G HA (SEQ ID NO: 1) (Molecular weight increase: 17)
Stable isotope labeled peptide C
VNP F R P GDSEPPPA P GAQRAQ (SEQ ID NO: 5) (molecular weight increment: 22)
Stable isotope labeled peptide D
SETESR G SES G IFTNTKESSSHHP G I A EFPSR G (SEQ ID NO: 3) (Molecular weight increase: 16)

50 μmol, 2.5 fmol and 25 fmol of stable isotope-labeled peptides A2, C2 and D2 were added to 1 μl of the serum of the colon cancer patient, respectively. Subsequently, the colon cancer marker polypeptide component was concentrated in the same manner as in Example 1, and the polypeptide component was fractionated into 60 fractions by reverse phase HPLC in the same manner as in Example 2. After concentrating the fractions containing the polypeptide markers A, C and D, MALDI-TOF-MS analysis was performed. The analysis data is shown in FIG. From data A, polypeptide marker A serum concentration is about 120 ng / ml, data C to polypeptide marker C is about 4 ng / ml, and data D to polypeptide marker D is about 60 ng / ml. understood.

(Example 7) Evaluation of diagnostic applicability of three types of polypeptide markers A, C and D in colorectal cancer patient serum Peptide marker A in the serum of 5 colorectal cancer patients before and after colorectal cancer extraction surgery, The colorectal cancer peptide marker of the present invention was re-evaluated by quantitative analysis of the abundance of C and D. For the evaluation, the same stable isotope-labeled peptide and analysis procedure as in Example 6 were used. The only difference is that 25 fmol, 5 fmol and 50 fmol of stable isotope labeled peptides A2, C2 and D2 are added per 1 ml of the serum.

Table 2 below shows the results of quantitative analysis of the concentrations of polypeptide markers A, C and D contained in the sera of five patients before and after colorectal cancer removal surgery. As shown in Table 2, in patients 1 to 5, the concentrations of polypeptide markers A, C and D were high before the operation, and the concentrations were clearly decreased after the operation. From this result, the abundance of the polypeptide marker of the present invention in serum correlates with the presence or absence (or abundance) of colorectal cancer in the body, and is considered to be sufficiently effective as a colorectal cancer diagnostic marker. .

The cleavage pattern of a 4-residue peptide in tandem MS (MS / MS) mass spectrometry is shown. It is a flowchart which shows the procedure of colon cancer marker polypeptide search of this invention. It is a flowchart which shows the concentration procedure of the colon cancer marker polypeptide component contained in serum. This concentration method is called K method. The analysis conditions when the polypeptide concentrated solution obtained from serum in Example 2 is fractionated by reverse phase HPLC are shown.

The fraction of the polypeptide obtained by reverse phase HPLC in Example 3 is shown. FIG. 4 shows the results of MALDI-TOF-MS analysis of each fraction of the polypeptide obtained by reverse phase HPLC in Example 3, and the polypeptides of healthy subject serum N1 to N8 and colon cancer patient sera P1 to P8 in each fraction. The presence of The white circle in FIGS. 6-9 shows the polypeptide which exists in healthy subject serum N1-N8, and a gray circle shows polypeptide which exists in colon cancer patient serum P1-P8. In Example 3, the presence of polypeptides of healthy subject serum N1 to N8 and colon cancer patient sera P1 to P8 in each fraction of polypeptides obtained by reverse phase HPLC is shown. In Example 3, the presence of polypeptides of healthy subject serum N1 to N8 and colon cancer patient sera P1 to P8 in each fraction of polypeptides obtained by reverse phase HPLC is shown. In Example 3, the presence of polypeptides of healthy subject serum N1 to N8 and colon cancer patient sera P1 to P8 in each fraction of polypeptides obtained by reverse phase HPLC is shown.

According to the spectrum of the MALDI-TOF-MS method, a part of the spectrum of fraction No. 6 (m / z = 2092, and 2163), a part of the spectrum of fraction No. 15 (m / z = 2188), and This is data showing that a peptide specific to serum of colon cancer patients is detected in a part of the spectrum of fraction No. 18 (m / z = 3505). It is the graph which showed the average spectrum of MS spectrum (Healthy person serum N1-N8, colon cancer patient serum P1-P8) of each colon cancer marker polypeptide. The graph (left) showing the mean and standard deviation of the MS peak intensity of each colon cancer marker polypeptide and the P value (right) obtained from the MS peak intensity of each colon cancer marker polypeptide are shown. For m / z = 2092 (A), m / z = 2163 (B), m / z = 2188 (C), m / z = 3505 (D), they were 0.0013, 0.0021,0.0006, 0.0047, respectively. . It is data which shows the identification result of colon cancer marker polypeptide A. From this result, it was revealed that the colon cancer peptide marker A is a polypeptide corresponding to SEQ ID NO: 1.

It is data which shows the identification result of colon cancer marker polypeptide C. From this result, it was revealed that the colon cancer peptide marker C is a polypeptide corresponding to SEQ ID NO: 5. It is data which shows the identification result of colon cancer marker polypeptide D. From this result, it was revealed that the colon cancer peptide marker D is a polypeptide corresponding to SEQ ID NO: 3. The flowchart of the quantitative analysis of marker polypeptide A using a stable isotope labeled polypeptide is shown. It is a conceptual diagram of a stable isotope labeled polypeptide. ^The results of quantitative analysis of the concentration of peptide marker A contained in the serum of the same patient before and after colorectal cancer excision surgery using ¹³ C-labeled peptide marker A are shown. FIG. 19 shows the results of tandem MS measurement (MS / MS measurement) for identification of colorectal cancer polypeptide markers, and is data that confirms that the identified peptide is actually the peptide. 19 (a) and (b) are MS / MS spectra of peptides B and A, respectively. Furthermore, the result of having interpreted the MS / MS spectrum measurement result is shown. Figures 13-15 are tables summarizing the interpretation of this data for peptides A, C and D. FIG. 20 shows the results of tandem MS measurement (MS / MS measurement) for identification of colorectal cancer polypeptide markers, which is data supporting that the identified peptide is actually the peptide. 20 (c) and (d) are MS / MS spectra of peptides C and D, respectively. Furthermore, the result of having interpreted the MS / MS spectrum measurement result is shown. Figures 13-15 are tables summarizing the interpretation of this data for peptides A, C and D. (A) to (c) in FIG. 21 show the MS / MS spectrum measurement results (black spectrum, lower panel) of the peptides chemically synthesized based on the amino acid sequences obtained from the identification results of peptides A, C and D, respectively. FIG. 19 shows a comparison of the MS / MS spectra (gray spectrum, upper part) of (a) of FIG. 19 and (c) and (d) of FIG. This figure shows that the peptide of the present invention is definitely the previously identified amino acid sequence. FIG. 22 is MS / MS spectrum data showing the abundance of colon cancer polypeptide markers A, C and D in the serum of one colon cancer patient.

(SEQ ID NO: 1) This shows the amino acid sequence of colorectal cancer marker polypeptide A of the present invention. The polypeptide is a partial peptide of residues 605 to 624 of the fibrinogen α chain.
(SEQ ID NO: 2) This shows the amino acid sequence of colorectal cancer marker polypeptide B of the present invention. The polypeptide is a partial peptide of residues 604 to 624 of the fibrinogen α chain.
(SEQ ID NO: 3) This shows the amino acid sequence of colorectal cancer marker polypeptide D of the present invention. The polypeptide is a partial peptide of residues 542 to 574 of the fibrinogen α chain.
(SEQ ID NO: 4) This is an amino acid sequence of a fibrinogen α chain including the amino acid sequences of the colon cancer marker polypeptides A, B and D of the present invention. The name of the corresponding gene is FGA.
(SEQ ID NO: 5) This shows the amino acid sequence of colorectal cancer marker polypeptide C of the present invention. The polypeptide is a partial peptide of residues 36 to 56 of dixin (Zyxin).
(SEQ ID NO: 6) This is an amino acid sequence of dixin, including the amino acid sequence of colorectal cancer marker polypeptide C of the present invention. The name of the corresponding gene is ZYX.

(SEQ ID NO: 7) This shows the nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide A of the present invention.
(SEQ ID NO: 8) This shows the nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide B of the present invention.
(SEQ ID NO: 9) This shows the nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide D of the present invention.
(SEQ ID NO: 10) This shows the nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide C of the present invention.

(SEQ ID NO: 11) This shows the mixed base nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide A of the present invention.
(SEQ ID NO: 12) This shows the mixed base nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide B of the present invention.
(SEQ ID NO: 13) This shows the mixed base nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide D of the present invention.
(SEQ ID NO: 14) This shows the mixed base nucleotide sequence of cDNA encoding the amino acid sequence of colorectal cancer marker polypeptide C of the present invention.

Claims (9)

  A method for detecting a colorectal cancer marker-positive person, comprising at least one selected from the group of peptides having an amino acid sequence consisting of SEQ ID NOs: 1 to 3 and 5, comprising a treatment of removing carrier protein from serum or plasma obtained from a subject A method for detecting a polypeptide of a species.   The method according to claim 1, wherein serum or plasma obtained from a subject is subjected to mass spectrometry.   A method for detecting a colorectal cancer marker-positive person, the method comprising detecting at least one polypeptide selected from the group of peptides having an amino acid sequence consisting of SEQ ID NOs: 1 to 3 and 5, wherein the peptide is an isotope element. The method according to claim 1 or 2, which is used as a labeled comparative peptide.   The method for detecting a prone to colorectal cancer marker according to claim 3, wherein the isotope is an oxygen atom, a carbon atom, a hydrogen atom or a nitrogen atom.   A method for obtaining data for diagnosing a prone colorectal cancer marker, comprising a process of removing carrier protein from serum or plasma obtained from a subject, comprising a peptide having an amino acid sequence consisting of SEQ ID NOs: 1 to 3 and 5 Detecting at least one polypeptide selected from the group.   6. The method for acquiring data for diagnosis of a colorectal cancer marker positive person according to claim 5, wherein the subject is a human having colorectal cancer or a human who has undergone colorectal cancer removal surgery.   The method for obtaining data for diagnosis of a colorectal cancer marker positive person according to claim 5 or 6, wherein serum or plasma obtained from a subject is subjected to mass spectrometry.   A method for obtaining data for diagnosis of a colorectal cancer marker-positive person, wherein the method detects at least one polypeptide selected from the group of peptides having an amino acid sequence consisting of SEQ ID NOs: 1 to 3 and 5. The method according to any one of claims 5 to 7, wherein the peptide is used as a comparative peptide labeled with an isotope element.   The method for acquiring data for diagnosis of a colorectal cancer marker positive person according to claim 8, wherein the isotope is an oxygen atom, a carbon atom, a hydrogen atom or a nitrogen atom.

Images