METHOD FOR DIAGNOSIS OF COLORECTAL CANCER USING MASS SPECTROMETRY OF N-GLYCANS

Abstract

The present invention relates to a method of diagnosing colorectal cancer by detection of glycan changes, and more particularly to a method of diagnosing colorectal cancer using mass spectrometry, in which, when specific glycan structures increase, decrease or significantly change due to a change in N-linked glycosylation of a colorectal cancer patient-derived glycoprotein, as detected by mass spectrometry, the glycan structures are selected as diagnostic markers.

Claims

1.-13. (canceled)

14. A method for diagnosing a colorectal cancer, comprising: (a) measuring a content of at least one biomarker selected from the group consisting of Hex5-HexNAc2 glycan (1234.4 m/z), Hex6-HexNAc2 glycan (1396.5 m/z), Hex7-HexNAc2 glycan (1558.5 m/z), Hex8-HexNAc2 glycan (1720.6 m/z), Hex9-HexNAc2 glycan (1882.7 m/z), Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc3 glycan (1437.5 m/z), Hex3-HexNAc4-Fuc1 glycan (1462.5 m/z), Hex4-HexNAc4-Fuc1 glycan (1624.6 m/z), Hex4-HexNAc4-NeuAc1 glycan (1769.6 m/z), Hex4-HexNAc4-Fuc1-NeuAc1 glycan (1915.7 m/z), Hex5-HexNAc4 glycan (1640.6 m/z), Hex5-HexNAc4-Fuc1 glycan (1786.7 m/z), Hex5-HexNAc4-NeuAc1 glycan (1931.7 m/z), Hex5-HexNAc4-Fuc1-NeuAc1 glycan (2077.7 m/z), Hex6-HexNAc4 glycan (1802.7 m/z), Hex3-HexNAc5-Fuc1 glycan (1665.6 m/z), Hex4-HexNAc5-Fuc1 glycan (1827.7 m/z), Hex5-HexNAc5-Fuc1 glycan (1989.7 m/z), Hex5-HexNAc5-NeuAc1 glycan (2134.8 m/z), Hex5-HexNAc5-Fuc1-NeuAc1 glycan (2280.8 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), Hex6-HexNAc5-Fuc1 glycan (2151.8 m/z), Hex6-HexNAc5-Fuc2 glycan (2297.9 m/z), Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z), Hex6-HexNAc5-Fuc1-NeuAc1 glycan (2442.9 m/z), Hex7-HexNAc6 glycan (2370.9 m/z), Hex7-HexNAc6-Fuc1 glycan (2516.9 m/z), and Hex7-HexNAc6-Fuc1-NeuAc1 glycan (2808.0 m/z), from a subject-derived blood sample; (b) diagnosing the subject to have colorectal cancer, when the content of the biomarker derived from the subject-derived blood sample has a T-test p-value of 0.05 or less, or an AUC (Area under the ROC curve) value of 0.7 or higher, compared to that derived from a normal blood sample.

15. The method of claim 14, wherein the colorectal cancer biomarker is at least one selected from the group consisting Hex5-HexNAc2 glycan (1234.4 m/z), Hex6-HexNAc2 glycan (1396.5 m/z), Hex7-HexNAc2 glycan (1558.5 m/z), Hex8-HexNAc2 glycan (1720.6 m/z), and Hex9-HexNAc2 glycan (1882.7 m/z).

16. The method of claim 14, wherein the colorectal cancer biomarker is at least one selected from the group consisting Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), and Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z).

17. The method of claim 14, wherein the content of a biomarker in step (a) is analyzed by LC/MS analysis.

18. The method of claim 14, wherein the LC/MS analysis is nano-LC chip/Q-TOF mass spectrometry (MS).

19. The method of claim 14, wherein the blood sample is whole blood, serum, or plasma.

Description

DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1(a) shows the profile of total glycans analyzed by mass spectrometry of haptoglobins purified from the sera of normal persons and colorectal cancer patients, and is a graph showing the results of identifying glycan marker candidates showing a significant difference. Purified haptoglobins were treated with PNGase F to isolate only N-glycans, and then the N-glycans of haptoglobins derived from a normal control group and a colorectal cancer patient group were profiled by LC-MS. Each of glycan structures is shown as relative abundance, and all structures within the upper 95% of the total structures are shown. FIG. 1(b) shows three glycan structures showing the most significant difference among total glycans. High-mannose structures (5200, 6200 and 7200) were all identified to be potent colorectal cancer biomarker candidates showing an AUC 0.9 or more and a sensitivity and specificity of 80% or more.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] Hereinafter, the configuration of the present invention will be described in more detail by way of Examples. However, it will be obvious to those skilled in the art that the scope of the present invention is not limited to only these Examples. In examples of the present invention, haptoglobin was used as an example of glycoprotein. However, it will be obvious to those skilled in the art that glycosylation-related enzymes are not glycosylated for certain proteins and that haptoglobin is described as an example of typical glycoprotein.

[0023] Materials and Other Reagents Anti-human beta-haptoglobin antibody was purchased from Dako (Carpinteria, Calif.). PNGase F (Peptide N-glycosidase F) was purchased from New England Biolabs (MA, USA). Graphitized carbon cartridges were purchased from Grace Davison Discovery Sciences (IL, USA). Mass spectrometry calculation (ESI-TOF Calibrant calibrant Mix mix G1969-85000) was performed using a product obtained from Agilent Technologies (CA, USA). All the chemicals used were of analytical grade or better.

[0024] Serum Samples from Colorectal Cancer Patients and Normal Persons

[0025] Serum samples were obtained from Chungnam National University Hospital (Korea), a member of the National Biobank of Korea. Clinical information about 20 colorectal cancer patients and 20 normal persons is summarized in Tables 1 and 2. The patients were biopsied and diagnosed by pathologists. This study was approved by the KAIST Institutional Review Board and was conducted under the consent of the participated normal persons and colorectal cancer patients.

[0026] Purification of Haptoglobin from Human Serum

[0027] Using anti-haptoglobin antibody, an anti-haptoglobin affinity column was prepared, and purification was performed. 500 l of serum was obtained from each of 20 colorectal cancer patients and 20 normal persons and diluted in 4 ml, of PBS (phosphate-buffered saline, 10 mM phosphate buffer/2.7 mM KCl/137 mM NaCl, pH 7.4), and each of the dilutions was applied to the anti-haptoglobin affinity column and incubated in a rotating agitator at room temperature for 2 hours. Unbound materials were removed by washing the column with 30 ml, of PBS, and haptoglobin was eluted with elution buffer (0.1 M glycine/0.5 M NaCl, pH 2.8), and then fractionated into tubes containing neutralization buffer. The eluent was concentrated, and then centrifuged using a centrifugal filter (molecular weight cut-off: 10,000, Amicon Ultra, Millipore) to remove the surfactant, and then assayed for haptoglobin by a Quant-iT Assay Kit, after which it was subjected to 12.5% SDS-PAGE and Quant-iT Assay Kit (Invitrogen, Carlsbad, Calif.) and Coomassie blue staining. The samples were freeze-dried, and stored at 80 C. until use in analysis.

[0028] N-Glycan Isolation Using Enzyme

[0029] PNGase F (peptide N-glycosidase F; 500,000 unit/ml) derived from Flavobacterium meningosepticum was purchased from New England BioLabs (Ipswich, Mass.). To isolate glycans from protein by use of enzyme, 50 l of the haptoglobin obtained in the above Example was dissolved in digestion buffer (pH 7.5, 100 mM ammonium bicarbonate, 5 mM DTT), and heated in boiling water for 2 minutes to denature the protein. After cooling at room temperature, 2 l of PNGase F (1,000 units) was added thereto, and the mixture was incubated in a water bath at 37 C. for 16 hours.

[0030] 400 l of cold ethanol was added to the incubated mixture to precipitate the peptide and the protein.

[0031] The resulting solution was frozen at 40 C. for 60 minutes, and then centrifuged at 14,000 rpm and 4 C. for 20 minutes. Next, for each sample, 400 l of the supernatant was collected, and ethanol contained in the supernatant was completely dried.

[0032] Thereafter, 1 ml of water was added to each sample, followed by intensive stirring, thereby preparing glycan-containing samples for purification.

[0033] Glycan Purification

[0034] Each of the glycan-containing samples isolated by PNGase F was purified by a graphitized carbon cartridge SPE (PGC-SPE; packing amount: 150 mg; cartridge volume: 3 ml). The PGC SPE cartridge was obtained from Alltech (Deerfield, Ill.). Prior to use, the cartridge was washed with 6 ml of ultrapure water, and washed with 6 ml of 80% (v/v) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), followed by washing with 6 ml of ultrapure water. The glycan-containing sample was placed in the PGC cartridge, and a several-fold volume of ultrapure water was allowed to flow through the cartridge at a rate of 1 ml/min to remove salts. N-glycans were eluted sequentially with 10% (v/v) acetonitrile, 20% (v/v) acetonitrile, and 40% (v/v) acetonitrile plus 0.05% (v/v) TFA. Each of the fractions was collected and dried with a centrifugal evaporator. The fractions were dissolved in ultrapure water before mass spectrometry.

[0035] Chip-Based Nano-LC/MS and MS/MS

[0036] Nano-LC separation was performed according to conventional technology. The N-glycan fractions for each sample were combined with each other, and 2.0 l (corresponding to 800 ng of haptoglobin) was loaded onto a nano-LC column (Agilent Technologies) having a chip placed thereon by an autosampler. The nano-LC column consists of an enrichment column (90.075 mm I.D.) and an analytical column (430.075 mm I.D.), both packed with 5 m porous graphitized carbon as the stationary phase. A rapid glycan elution gradient was delivered at a rate of 0.3 l/min using solutions of (A) 3.0% acetonitrile and 0.1% formic acid (v/v) in water, and (B) 90.0% acetonitrile and 0.1% formic acid (v/v) in water, ramping from 6% to 100% B solution over 20 minutes. Remaining non-glycan compounds were flushed out with 100% B solution prior to re-equilibration. After chromatographic separation, glycans were ionized by a chip-integrated nano-ESI spray tip and analyzed by a Q-TOF mass analyzer (Model 6540, Agilent Technologies) according to conventional technology. Calibrant molecules (ESI-TOF Calibrant Mix G1969-85000, Agilent Technologies) were injected directly into an electrospray mass spectrometer to make internal mass measurement possible. MS spectra were acquired in positive ionization mode over a mass range of m/z 500-2000 with an acquisition time of 2 seconds per spectrum. MS/MS spectra were acquired in positive ionization mode over a mass range of m/z 100-3000 with an acquisition time of 1.5 seconds per spectrum. Following an MS scan, precursor compounds were automatically selected for MS/MS analysis by the acquisition software based on ion abundance and charge state (z=2 or 3) and isolated in the quadrupole with a mass bandpass FWHM (full width at half maximum) of 1.3 m/z. Collision energies for CID fragmentation were calculated for each precursor compound based on the following formula:

V.sub.collision=1.8V{(m/z)/100 Da}4.8V

wherein V.sub.collision is the potential difference applied across the collision cell to accelerate and fragment the precursor. Raw LC/MS date was processed using the Molecular Feature Extractor algorithm included in the MassHunter Qualitative Analysis software (version B.04.00 SP2, Agilent Technologies). MS peaks were filtered with a signal-to-noise ratio of 5.0 and deconvoluted to create a list of compound mass, ion abundance and retention time.

[0037] Identification of N-Glycans by Accurate Mass

[0038] The compounds identified by nano-LC/MS were matched by accurate mass to a glycan database that covers all possible complex, hybrid, and high-mannose glycan compositions based on known biological synthesis pathways and glycosylation patterns. Deconvoluted mass of each ECC peak were compared against theoretical glycan mass using a mass error tolerance of 20 ppm. As the sample set originated from human serum, only glycan compositions containing hexose, HexNAc (N-acetylhexosamine), fucose and NeuAc (N-acetylneuraminic acid) were considered. Using T-test p-value analysis, receiver-operating characteristic (ROC) curve and AUC (Area under the ROC curve), N-glycans extracted from each sample were comparatively analyzed.

[0039] Results 1: Analysis of Colorectal Cancer-Specific N-Glycans of Haptoglobin

[0040] Detailed glycosylation patterns of the blood glycoprotein haptoglobin were analyzed by a chip-based nano-LC/TOF-MS (Chip/TOF) system. This system can identify the heterogeneity of glycans having different connection or antennary structures, and can provide higher sensitivity than MALDI-MS and conventional LC/MS, because of additional advantages such as the provision of low energy ion, large dynamic range and unmatched retention time reproducibility. In the present invention, the N-glycans of haptoglobins derived from the sera of normal persons and patients (n=40) were analyzed twice by nano-LC/MS. Only the N-glycans of haptoglobins were separated by PNGase F treatment, and then the N-glycans of haptoglobins derived from the normal control group and the colorectal cancer patient group were compared with one another by chip-based nano-/TOF-MS (Chip/TOF). All structures within the upper 95% of total N-glycan structures found in each sample were used, and quantitative values were compared with one another. Among high-mannose structures of the N-glycan structures, Hex5-HexNAc2 (5200 glycan structure) showing a mass value of 1234.43, Hex6-HexNAc2 (6200 glycan structure) showing a mass value of 1396.48, and Hex7-HexNAc2 (7200 glycan structure) showing a mass value of 1558.54, etc., showed an AUC value of 0.90 or higher. Furthermore, among several glycan structures showing a significant difference between the normal control group and the colorectal cancer patient group, Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), and Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z) structures in addition to high-mannose structures showed a difference between the colorectal cancer sample and the normal control group (P<0.01), but showed no difference between a gastric cancer patient group and the normal control group.

[0041] FIG. 1 shows the profile of total glycans analyzed by mass spectrometry of haptoglobins purified from the sera of normal persons and colorectal cancer patients, and is a graph showing the results of identifying glycan marker candidates showing a significant difference from the date. FIG. 1A shows the results obtained by treating purified haptoglobins with PNgase F to separate only N-glycans, and then profiling the N-glycans of haptoglobins, derived from a normal control group and a colorectal cancer patient group, by LC-MS. Each of N-glycan structures is shown as relative abundance, and all structures within the upper 95% of the total structures are shown. FIG. 1B shows three N-glycan structures showing the most significant difference among total N-glycans. High-mannose structures (5200, 6200 and 7200) were all identified to be potent colorectal cancer biomarker candidates showing an AUC 0.9 or higher and a sensitivity and specificity of 80% or higher.

[0042] Tables 1 and 2 show summary information about a total of 40 normal persons and colorectal cancer patients (20 normal persons and 20 colorectal cancer patients). Serum samples were obtained from Chungnam National University Hospital (Korea), a member of the National Biobank of Korea. The patients were biopsied and diagnosed by pathologists.

[0043] Tables 3 and 4 show a list of N-glycan structures showing a sensitivity corresponding to a p value of 0.05 or less between the normal control group and the colorectal cancer patient group, among N-glycan structures separated from the haptoglobins identified by nano LC chip/Q-TOF MS spectrometry. For example, N-chain structures can be identified based on a retention time library, and the amounts of all haptoglobin-derived N-chain structures can be determined. In Tables 3 and 4, glycan structures are classified into high-mannose structures and antennary structures, based on the results of mass (MS) mass spectrometry. Particularly, it was shown that several high-mannose structures (Hex5-HexNAc2 glycan (1234.4 m/z), Hex6-HexNAc2 glycan (1396.5 m/z), Hex7-HexNAc2 glycan (1558.5 m/z)) showed an AUC of 0.9 or higher, and thus could accurately distinguish the colorectal cancer patients from the normal persons. In addition, a Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), and Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z) structures showed a difference between the colorectal cancer samples and the normal control group (P<0.01), but showed no difference between gastric cancer patient samples and the normal control groups, indicating that it is a colorectal cancer-specific biomarker candidate distinguishable from gastric cancer.

[0044] The present invention uses body fluids such as serum as samples, and particularly, uses haptoglobins that are glycoproteins present in serum in large amounts. Thus, the present invention can be easily applied to in vitro diagnostic technology. Colorectal cancer-related biomarkers currently approved by the FDA include CEA protein, but the CEA protein has a limitation in that it shows a detection rate of about 70%.

[0045] The present invention can provide a method of diagnosing colorectal cancer based on the difference in expression of specific N-glycan structures (including high-mannose structures) between colorectal cancer patients and normal persons by analyzing N-glycan structures, which change in colorectal cancer patients compared to those in normal persons, by mass spectrometry. Glycan biomarkers, including high-mannose structures, identified by mass spectrometry, are listed in Tables 3 and 4. These glycan structures are biomarkers having a significance of p=0.05 or less.

[0046] The present invention can be applied for the development of a new method for diagnosis of colorectal cancer, a composition for diagnosis of colorectal cancer, and a kit for diagnosis of colorectal cancer.

TABLE-US-00001 TABLE 1 Case no. Classification Age Sex Location ClinicalTNM Stage cc1 Colon Cancer 71 F ascending T2N0M0 I Cc2 Colon Cancer 51 F sigmoid T3N0M0 II A Cc3 Colon Cancer 70 F rectum T3N0M0 II A Cc4 Colon Cancer 54 F descending T2N0M0 I Cc5 Colon Cancer 62 F sigmoid T3N0M0 II A Cc6 Colon Cancer 81 M ascending T3N0M0 II A Cc7 Colon Cancer 68 F ascending T1N0M0 I Cc8 Colon Cancer 65 M rectum T3N0M0 II A Cc9 Colon Cancer 73 M rectum T3N1bM0 III B Cc10 Colon Cancer 64 F ascending T3N0M0 II A cc11 Colon Cancer 73 F rectosigmoid T3N0M0 II A cc12 Colon Cancer 76 F rectum T0N0M0 III B cc13 Colon Cancer 69 F sigmoid TisN0M0 0 cc14 Colon Cancer 62 M sigmoid T3N0M0 II A cc15 Colon Cancer 64 M ascending T2N0M0 I cc16 Colon Cancer 61 M rectum T2N0M0 I cc17 Colon Cancer 70 F ascending T2N0M0 I cc18 Colon Cancer 66 F sigmoid T3N1M1 IV cc19 Colon Cancer 68 F sigmoid TN0M0 I cc20 Colon Cancer 70 F rectum T2N0M0 I

TABLE-US-00002 TABLE 2 Case no. Classification Age Sex n1 normal 34 M n2 normal 31 M n3 normal 31 M n4 normal 30 M n5 normal 53 F n6 normal 49 F n7 normal 49 F n8 normal 51 F n9 normal 49 F n10 normal 61 F n11 normal 61 F n12 normal 60 M n13 normal 34 F n14 normal 52 F n15 normal 51 M n16 normal 59 M n17 normal 47 F n18 normal 63 F n19 normal 46 M n20 normal 47 M

TABLE-US-00003 TABLE 3 Relative abundance(%) Composition Colon GlycanMass/Da Hex HexNAc Fuc NeuAc Normal Cancer t-Test AUC High Mannose 1234.43 5 2 0 0 0.58 0.19 0.000555 0.91 1396.48 6 2 0 0 0.40 0.11 0.000108 0.91 1558.54 7 2 0 0 0.29 0.07 0.000377 0.94 1720.59 8 2 0 0 0.27 0.08 0.008656 0.87 1882.65 9 2 0 0 0.10 0.03 0.000204 0.83 Mono, Bi-antennary 1566.56 4 3 0 1 3.4865 2.065 0.009169 0.77 1437.50 5 3 0 0 0.2468 0.1444 0.003803 0.75 1462.54 3 4 1 0 0.2645 0.1235 0.006786 0.74 1624.60 4 4 1 0 0.5336 0.2776 0.010488 0.72 1769.64 4 4 0 1 1.3232 0.8425 0.025722 0.76 1915.71 4 4 1 1 0.1402 0.0325 0.001757 0.83 1640.59 5 4 0 0 14.3 21.967 0.004744 0.77 1786.65 5 4 1 0 0.7582 1.2421 0.021130 0.65 1931.69 5 4 0 1 39.632 27.961 0.018485 0.77 2077.74 5 4 1 1 1.8768 0.9942 0.000309 0.85 1802.65 6 4 0 0 0.0261 0.0975 0.000467 0.72

TABLE-US-00004 TABLE 4 Relative abundance(%) Composition Colon GlycanMass/Da Hex HexNAc Fuc NeuAc Normal Cancer t-Test AUC Tri-antennary 1665.63 3 5 1 0 0.1043 0.0532 0.012200 0.69 1827.65 4 5 1 0 0.2608 0.134 0.007611 0.77 1989.73 5 5 1 0 0.4789 0.2619 0.000844 0.8 2134.76 5 5 0 1 0.7312 0.4441 0.000218 0.86 2280.82 5 5 1 1 1.8226 0.6526 0.000262 0.9 2571.92 5 5 1 2 0.3514 0.0355 0.000710 0.86 2005.72 6 5 0 0 6.7373 13.096 0.002537 0.8 2151.77 6 5 1 0 0.4991 2.2004 0.000539 0.79 2297.85 6 5 2 0 0.0269 0.1298 0.015538 0.6 2587.93 6 5 0 2 0.082 0.0206 0.003398 0.77 2442.88 6 5 1 1 1.0125 1.6435 0.012092 0.72 Tetra-antennary 2370.85 7 6 0 0 0.9597 2.1479 0.001962 0.75 2516.91 7 6 1 0 0.0566 0.4507 0.003370 0.74 2808.00 7 6 1 1 0.089 0.2639 0.005788 0.73

[0047] Haptoglobin is one of highly abundant glycoproteins, and is an acute phase protein that increases in the progression of various diseases such as inflammation and tumors. It is known that haptoglobin has four N-glycosylation sites at asparagines 184, 207, 211 and 241 and has one O-glycosylation site. A particular glycosylation type and a particular glycosylation site, which provides glycan changes that are distinguished between colorectal cancer patients and normal persons, are not known.

[0048] The present inventors performed the purification of serum-derived haptoglobin by anti-haptoglobin antibody affinity chromatography.

[0049] The present inventors determined an exact glycosylation state by chip-based nano-LC/TOF-MS (Chip/TOF) spectrometry following immune affinity chromatography purification. Because LC-MS causes increased sensitivity and less ion fragmentation compared to MALDI-MS, the present inventors could successfully demonstrate the detailed glycan structures of haptoglobins. In conclusion, modified N-glycans were detected in haptoglobins derived from colorectal cancer patients.

[0050] Several glycan structures showing a significant difference between a normal control group and a colorectal cancer patient group could be found by glycan structure profiling. Various N-glycan structures, including high-mannose structures, showed a significant difference in relative amount between the normal control group and the colorectal cancer patient group (Tables 3 and 4).

[0051] The present inventors have found the difference in high-mannose structures between the normal control group and the colorectal cancer patient group. Interestingly, this difference in high-mannose structures was observed by chip-based nano-LC/TOF-MS (Chip/TOF), because the use of this method could classify glycan structures with high sensitivity. This fact demonstrates that the high-sensitivity mass spectrometry method can be effectively used for diagnosis of cancer by use of biomarkers. Such results suggest that the abnormal glycan structures obtained in the present invention are useful glycan biomarkers that can replace current nonspecific colorectal cancer markers.

[0052] The present invention is related to a method for analyzing a colorectal cancer biomarker comprises: [0053] (a) isolating a haptoglobin from a subject-derived blood sample; [0054] (b) isolating a N-glycan from the isolated haptoglobin; [0055] (c) analyzing mass of the isolated N-glycan by LC/MS analysis; and [0056] (d) determining the structure and composition of the N-glycan, and performing quantitative profiling of the N-glycan based on the results of the LC/MS analysis.

[0057] In addition, the N-glycan in step (d) may be at least one selected from the group consisting of [0058] Hex5-HexNAc2 glycan (1234.4 m/z), [0059] Hex6-HexNAc2 glycan (1396.5 m/z), [0060] Hex7-HexNAc2 glycan (1558.5 m/z), [0061] Hex8-HexNAc2 glycan (1720.6 m/z), [0062] Hex9-HexNAc2 glycan (1882.7 m/z), [0063] Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), [0064] Hex5-HexNAc3 glycan (1437.5 m/z), [0065] Hex3-HexNAc4-Fuc1 glycan (1462.5 m/z), [0066] Hex4-HexNAc4-Fuc1 glycan (1624.6 m/z), [0067] Hex4-HexNAc4-NeuAc1 glycan (1769.6 m/z), [0068] Hex4-HexNAc4-Fuc1-NeuAc1 glycan (1915.7 m/z), [0069] Hex5-HexNAc4 glycan (1640.6 m/z), [0070] Hex5-HexNAc4-Fuc1 glycan (1786.7 m/z), [0071] Hex5-HexNAc4-NeuAc1 glycan (1931.7 m/z), [0072] Hex5-HexNAc4-Fuc1-NeuAc1 glycan (2077.7 m/z), [0073] Hex6-HexNAc4 glycan (1802.7 m/z), [0074] Hex3-HexNAc5-Fuc1 glycan (1665.6 m/z), [0075] Hex4-HexNAc5-Fuc1 glycan (1827.7 m/z), [0076] Hex5-HexNAc5-Fuc1 glycan (1989.7 m/z), [0077] Hex5-HexNAc5-NeuAc1 glycan (2134.8 m/z), [0078] Hex5-HexNAc5-Fuc1-NeuAc1 glycan (2280.8 m/z), [0079] Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), [0080] Hex6-HexNAc5 glycan (2005.7 m/z), [0081] Hex6-HexNAc5-Fuc1 glycan (2151.8 m/z), [0082] Hex6-HexNAc5-Fuc2 glycan (2297.9 m/z), [0083] Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z), [0084] Hex6-HexNAc5-Fuc1-NeuAc1 glycan (2442.9 m/z), [0085] Hex7-HexNAc6 glycan (2370.9 m/z), [0086] Hex7-HexNAc6-Fuc1 glycan (2516.9 m/z), and [0087] Hex7-HexNAc6-Fuc1-NeuAc1 glycan (2808.0 m/z).

[0088] In addition, the N-glycan in step (d) may be at least one selected from the group consisting of Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), and Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z).

[0089] The structures of the N-glycans show a difference between a colorectal cancer sample and a normal sample (P<0.01), but no difference between a gastric cancer sample and a normal sample.

[0090] In addition, the N-glycan in step (d) may be at least one selected from the group consisting of Hex5-HexNAc2 glycan (1234.4 m/z), Hex6-HexNAc2 glycan (1396.5 m/z), Hex7-HexNAc2 glycan (1558.5 m/z), Hex8-HexNAc2 glycan (1720.6 m/z), and Hex9-HexNAc2 glycan (1882.7 m/z).

[0091] In addition, the LC/MS analysis in step (c) may be nano-LC chip/Q-TOF mass spectrometry (MS).

[0092] In addition, the quantitative profiling in step (d) may be performed using at least one selected from the group consisting of T-test p-value analysis, ROC (Receiver-Operating Curve) analysis, and AUC (Area under the ROC curve) analysis

[0093] In addition, the blood sample may be whole blood, serum, or plasma.

[0094] The present invention is also related to a method for analyzing a colorectal cancer biomarker comprises: [0095] (a) isolating a haptoglobin from each of a subject-derived blood sample and a normal blood sample; [0096] (b) isolating N-glycans from each of the isolated haptoglobins; [0097] (c) analyzing mass of the isolated N-glycans by LC/MS analysis; [0098] (d) determining the structure and composition of the N-glycans, and performing quantitative profiling of the N-glycans based on the results of the LC/MS analysis; and [0099] (e) selecting the N-glycan derived from the subject-derived blood sample as the colorectal cancer biomarker, when the N-glycan derived from the subject-derived blood sample has either a T-test p-value of 0.05 or less compared to that of the N-glycan derived from the normal blood sample, or an AUC (Area under the ROC curve) value of 0.7 or higher.

[0100] In addition, the method for analyzing a colorectal cancer biomarker further comprise, after step (e), step (f) of determining the subject has colorectal cancer when the content of the subject sample-derived N-glycan which is selected as the colorectal cancer biomarker has a significant difference from the content of the normal blood sample-derived N-glycan.

[0101] In addition, the selected colorectal cancer biomarker in step (e) may be at least one selected from the group consisting of [0102] Hex5-HexNAc2 glycan (1234.4 m/z), [0103] Hex6-HexNAc2 glycan (1396.5 m/z), [0104] Hex7-HexNAc2 glycan (1558.5 m/z), [0105] Hex8-HexNAc2 glycan (1720.6 m/z), [0106] Hex9-HexNAc2 glycan (1882.7 m/z), [0107] Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), [0108] Hex5-HexNAc3 glycan (1437.5 m/z), [0109] Hex3-HexNAc4-Fuc1 glycan (1462.5 m/z), [0110] Hex4-HexNAc4-Fuc1 glycan (1624.6 m/z), [0111] Hex4-HexNAc4-NeuAc1 glycan (1769.6 m/z), [0112] Hex4-HexNAc4-Fuc1-NeuAc1 glycan (1915.7 m/z), [0113] Hex5-HexNAc4 glycan (1640.6 m/z), [0114] Hex5-HexNAc4-Fuc1 glycan (1786.7 m/z), [0115] Hex5-HexNAc4-NeuAc1 glycan (1931.7 m/z), [0116] Hex5-HexNAc4-Fuc1-NeuAc1 glycan (2077.7 m/z), [0117] Hex6-HexNAc4 glycan (1802.7 m/z), [0118] Hex3-HexNAc5-Fuc1 glycan (1665.6 m/z), [0119] Hex4-HexNAc5-Fuc1 glycan (1827.7 m/z), [0120] Hex5-HexNAc5-Fuc1 glycan (1989.7 m/z), [0121] Hex5-HexNAc5-NeuAc1 glycan (2134.8 m/z), [0122] Hex5-HexNAc5-Fuc1-NeuAc1 glycan (2280.8 m/z), [0123] Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), [0124] Hex6-HexNAc5 glycan (2005.7 m/z), [0125] Hex6-HexNAc5-Fuc1 glycan (2151.8 m/z), [0126] Hex6-HexNAc5-Fuc2 glycan (2297.9 m/z), [0127] Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z), [0128] Hex6-HexNAc5-Fuc1-NeuAc1 glycan (2442.9 m/z), [0129] Hex7-HexNAc6 glycan (2370.9 m/z), [0130] Hex7-HexNAc6-Fuc1 glycan (2516.9 m/z), and [0131] Hex7-HexNAc6-Fuc1-NeuAc1 glycan (2808.0 m/z).

[0132] In addition, the selected colorectal cancer biomarker in step (e) may be at least one selected from the group consisting Hex5-HexNAc2 glycan (1234.4 m/z), Hex6-HexNAc2 glycan (1396.5 m/z), Hex7-HexNAc2 glycan (1558.5 m/z), Hex8-HexNAc2 glycan (1720.6 m/z), and Hex9-HexNAc2 glycan (1882.7 m/z).

[0133] In addition, the selected colorectal cancer biomarker in step (e) may be at least one selected from the group consisting Hex4-HexNAc3-NeuAc1 glycan (1566.6 m/z), Hex5-HexNAc5-Fuc1-NeuAc2 glycan (2571.9 m/z), Hex6-HexNAc5 glycan (2005.7 m/z), and Hex6-HexNAc5-NeuAc2 glycan (2587.9 m/z).

[0134] In addition, the blood sample may be whole blood, serum, or plasma.

INDUSTRIAL APPLICABILITY

[0135] As described above, the present invention can be used for the diagnosis and prevention of colorectal cancer.