WO2016175393A1 - Markers for diagnosing angiostenosis and use thereof - Google Patents

Abstract

Provided are: a composition for diagnosing non-diabetic angiostenosis, containing a preparation measuring the level of one or more proteins selected from the group consisting of poly (ADP-ribose) polymerase 4 (PARP4), vitamin D-binding protein (VDB), and laminin subunit beta-1 (LAMB1); a composition for diagnosing diabetic angiostenosis, containing a preparation measuring the level of one or more proteins selected from the group consisting of interferon regulatory factor 7 (IRF7), a dedicator of cytokinesis protein 2 (DOCK2), and a hemoglobin subunit beta (HBB); a kit for diagnosing angiostenosis, containing the composition for diagnosing diabetic angiostenosis; a method for providing information for diagnosing non-diabetic angiostenosis; and a method for providing information for diagnosing non-diabetic angiostenosis. The present invention provides a composition for diagnosing angiostenosis, so as to measure and compare the changing protein expression levels in non-diabetic or diabetic angiostenosis patients, thereby enabling early diagnosis of angiostenosis and a meaningful prediction or an identification of the degree of the disease. In addition, the diagnostic composition of the present invention enables non-invasive diagnosis, thereby enabling simple and valid early stage diagnosis of angiostenosis by using blood and urine testing and the like.

Description

Markers for diagnosing vascular stenosis and uses thereof

The present invention is a composition for diagnosing non-diabetic vascular stenosis, a composition for diagnosing diabetic vascular stenosis, and a kit for diagnosing vascular stenosis, a method for providing information for diagnosing non-diabetic vascular stenosis, and diabetic vascular It provides a method of providing information for diagnosing stenosis disease.

Recently, the number of patients with metabolic syndrome is increasing rapidly due to urbanization of living environment and excessive nutrition. There is an increased risk of diabetes and obesity, a metabolic disease worldwide, and the prevalence of diabetes has begun to exceed 10% of the population.

According to the results of the 2005-2007 National Health and Nutrition Survey, 10% of the adult population is diabetic and more than 30% are obese, and the explosive increase in diabetes is a worldwide trend. In addition, the number of deaths from cerebrovascular disease, heart disease, diabetes mellitus, and hypertension disease exceeds that of cancer.

Cardiovascular disease is the leading cause of death in patients with diabetes. Myocardial infarction and cerebral infarction account for 40% of all diabetes deaths. In particular, diabetic cardiovascular disease, asymptomatic cases are often a lot of symptoms such as chest pain, dizziness, headaches, numbness and vascular stenosis is already in progress, so it is likely to die of severe disease when found. Therefore, early detection of vascular stenosis and prevention of disease are very important.

Currently used early detection of vascular stenosis is diagnostic methods that indirectly predict the degree of arteriosclerosis. Carotid artery Doppler ultrasound, pulse wave velocity, blood flow-dependent vasodilation response ( flow mediated dilatation). Recently developed and clinically applied multi-detector coronary computed tomography (MDCT) still has limitations such as the use of expensive contrast agents and accompanying functional tests.

It is urgent to develop a biomarker that can be diagnosed early in cardiovascular disease, which has been identified as the leading cause of death in diabetes and diabetes patients, the most important chronic diseases of Koreans. To date, some changes in serum proteins are known, such as a decrease in adiponectin secreted by adipocytes and an increase in CRP, TNF-α, IL-6, etc., which are involved in systemic inflammation such as atherosclerosis. There is no established biomarker that can reliably predict the extent and disease state of cardiovascular disease, particularly vascular stenosis, in diabetics.

Considering the prevalence of metabolic diseases such as obesity, diabetes and metabolic syndrome, which are explosively increasing in Korea, all of these disease groups are predictive of vascular stenosis as ischemic cardiovascular complications are the main cause of death. The excavation of the urgent situation is urgent.

In order to diagnose the progress of vascular stenosis early, the present inventors introduced a proteomic technique to discover biomarkers. In particular, there have been many reports on changes in inflammatory proteins related to diabetes, a high risk group of vascular stenosis, but no changes in total blood proteins between diabetes and vascular stenosis have been reported. By analyzing the whole blood protein in the patient group according to the proteomics technique, it was confirmed that the proteins that change according to the presence or absence of diabetes mellitus, and the degree of vascular stenosis can be used as a biomarker, through which vascular stenosis in diabetic patients or non-diabetic patients By confirming that the progress can be monitored, the present invention has been completed.

One object of the present invention is to provide a composition for diagnosing non-diabetic vascular stenosis disease.

Another object of the present invention is to provide a composition for diagnosing diabetic vascular stenosis disease.

Still another object of the present invention is to provide a kit for diagnosing a vascular stenosis disease, comprising the composition.

Still another object of the present invention is to provide a method of providing information for diagnosing non-diabetic vascular stenosis disease.

Still another object of the present invention is to provide a method of providing information for diagnosing diabetic vascular stenosis disease.

The present invention, by providing a composition for diagnosing vascular stenosis disease, by measuring and comparing the expression level of the protein expression changes in patients with non-diabetic or diabetic vascular stenosis disease, significant early diagnosis and disease degree of vascular stenosis disease significantly Can be predicted or identified.

In addition, the diagnostic composition of the present invention enables a non-invasive diagnosis and can make a simple and effective initial diagnosis of vascular stenosis disease by blood, urine test, and the like.

Figure 1 shows a protein with a significant difference in expression according to vascular stenosis in non-diabetic patients.

Figure 2 shows a protein with a significant difference in expression according to vascular stenosis in diabetic patients.

In order to achieve the above object, the present invention provides a composition for diagnosing vascular stenosis disease as one embodiment.

In one aspect, the composition for diagnosing vascular stenosis is poly (ADP-ribose) polymerase 4 (PARP4), vitamin D-binding protein (VDB) and laminin sub It may be a composition for diagnosing non-diabetic vascular stenosis disease, comprising an agent for measuring at least one protein level selected from the group consisting of unit beta-1 (Laminin subunit beta-1, LAMB1).

In the present invention, the term "vascular stenosis disease" is a disease in which blood vessels are narrowed, and when narrowing, blood flow is reduced and ischemia occurs in a site where a narrowed blood vessel is distributed, or blood clots or hardened fragments are formed in atherosclerosis with a rough surface. A detached embolus can develop, including, but not limited to, a disease that causes symptoms. In the case of the vascular stenosis disease of the present invention, as the endovascular thickening progresses, vascular narrowing may occur, and the elasticity of the vascular wall may be reduced to cause bleeding due to vascular rupture. In the present invention, the vascular stenosis disease may be stroke, atherosclerosis, in-stent restenosis, myocardial infarction, or atherosclerosis, but is not limited thereto.

As used herein, the term "diagnosis" refers to identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to determine whether the vascular stenosis disease, the diagnosis can be confirmed early in the vascular stenosis disease.

In the present invention, the poly (ADP-ribose) polymerase 4 (Poly (ADP-ribose) polymerase 4, PARP4) is a poly (ADP-ribosyl) transferase-like 1 protein that catalyzes the poly (ADP-ribosyl) ation reaction The present inventors have first identified the relationship between the vascular stenosis disease.

The vitamin D-binding protein (VDB) is known as VDB-globulin (group-specific component), belongs to the albumin gene family, and is known to bind vitamin D to deliver vitamin D to target tissues. In addition, the present inventors first identified the relationship with the vascular stenosis disease.

The laminin subunit beta-1 (LAMB1) is an extracellular matrix constituent that interacts with other extracellular matrix components and is known to be involved in cell adhesion, migration, and the like. The inventors first identified the relationship.

In another aspect, the composition for diagnosing vascular stenosis disease of the present invention is an interferon regulatory factor 7 (IRF7), a indicator of cytokinesis protein 2 (DOCK2), and hemoglobin sub It may be a composition for diagnosing diabetic vascular stenosis disease, including an agent for measuring the level of one or more proteins selected from the group consisting of unit beta (Hemoglobin subunit beta, HBB).

The interferon regulatory factor 7 (IRF7) belongs to the family of interferon regulatory transcription factors, and is known to activate the transcription of virus-induced cellular genes. First identified.

Dedicator of cytokinesis protein 2 (DOCK2) is known to be involved in intracellular signaling by activating Rac, a G protein, and related to vascular stenosis in diabetic patients. The inventors first identified.

The hemoglobin subunit beta (HBB) is known to interact with hemoglobin alpha 1 (HBA1), which forms hemoglobin A, and was first identified by the present inventors regarding its association with vascular stenosis in diabetic patients. .

In one embodiment of the present invention, the non-diabetic patients are classified into low risk group (0%), medium risk group (0-50%), high risk group (more than 50%) according to the degree of vascular stenosis, The patients were classified into low risk, medium risk, and high risk patients. Afterwards, blood samples were collected from these patients and the expression patterns of the proteins between the risk groups of vascular stenosis were observed using relative quantitative analysis using TMT (Tandem Mass Tag). 100 significant (p value <0.05) proteins were selected as primary biomarker candidates. In order to verify the selected primary biomarker candidate group, verification of the primary biomarker candidate groups was performed using 10 patient samples in each group. Finally, in the non-diabetic patient group, six proteins, PARP4, VDB, LAMB1, C4A, LBP, and APOC2 were significantly changed according to the risk of vascular stenosis, confirming that these proteins could be used as diagnostic markers for vascular stenosis. In addition, six proteins, such as IRF7, DOCK2, HBB, C4A, LBP, and APOC2, also change significantly according to the risk of vascular narrowing in diabetic patients. It was confirmed that it can be used as a marker that can be.

In particular, among the biomarkers identified in non-diabetic patients, PARP4 decreased the expression of PARP4 protein in the middle-risk group compared to the low-risk group, and C4A decreased the expression of C4A protein in the high-risk group compared to the low-risk group. The expression of APOC2 protein was increased in both the high risk group and the high risk group, compared to the low risk group, and the expression of VDB protein was decreased in both the high risk group and the low risk group, and LAMB1 was higher in the high risk group than the low risk group. In all, the expression of LAMB1 protein was decreased, and LBP increased the expression of LBP protein in the high risk group compared to the medium risk group.

In addition, among the biomarkers identified in diabetic patients, IRF7 increased the expression of IRF7 protein in both the high risk group and the low risk group, and DOCK2 increased the expression of DOCK2 protein in both the high risk group and the low risk group. APOC2 decreased the expression of APOC2 protein in both the high risk and high risk groups compared to the low risk group, HBB increased the expression of HBB protein in the high risk group compared to the low risk group, and LBP increased the expression of HBB. The expression of LBP protein was increased in all high risk groups, and the expression of C4A protein was increased in the high risk group compared to the medium risk group.

Accordingly, the PARP4, VDB, LAMB1, C4A, LBP, and APOC2 are all compared with the normal control group (diabetic and vascular stenosis), and the expression level of the protein is changed in the vascular stenosis. Because of its characteristics, it can be used as a marker for diagnosing non-diabetic vascular stenosis.

In addition, the IRF7, DOCK2, HBB, C4A, LBP, APOC2 are all compared to the control group (diabetic, but not vascular stenosis), the expression level of the protein is changed in individuals with diabetes and vascular stenosis disease Because of its characteristics, it can be used as a marker for diagnosing diabetic vascular stenosis.

As used herein, the term "marker" refers to a substance which can be diagnosed by distinguishing between a normal group and an individual having a vascular stenosis. Polypeptides, proteins, which show an increase or decrease in an individual having a vascular stenosis, And all organic biomolecules such as glycoproteins. In particular, the present invention may be a protein that is changed in an individual having a non-diabetic vascular stenosis or an individual having a diabetic vascular stenosis, but is not limited thereto.

The term "protein level measurement" used in the present invention is a process of confirming the presence and expression level of a marker protein for diagnosing vascular stenosis disease in a biological sample for diagnosing vascular stenosis disease. The amount of the protein can be confirmed using an antibody specifically binding to the marker protein, or the protein expression level itself can be measured without using the antibody.

The protein level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, radioimmunoassay, radioimmunoassay, oukteroni Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complementary Assay, 2-D Electrophoresis, Liquid Chromatography-Mass Spectrometry (LC-MS), Liquid Chromatography-Mass (LC-MS / MS) Spectrometry / Mass Spectrometry), Western Blot, and Enzyme linked immunosorbentassay (ELISA).

The agent for measuring the protein level for diagnosing non-diabetic vascular stenosis disease may include an antibody that specifically binds to a protein selected from PARP4, VDB, and LAMB1.

In addition, the agent for measuring the protein level for diagnosing diabetic vascular stenosis disease may include an antibody that specifically binds to a protein selected from IRF7, DOCK2, and HBB.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to at least one protein selected from PARP4, VDB, LAMB1, IRF7, DOCK2, and HBB, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Include. Generating antibodies can be readily prepared using techniques well known in the art.

The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. Functional fragments of antibody molecules mean fragments having at least antigen binding function, and include Fab, F (ab '), F (ab') 2 and Fv.

As another aspect, the present invention provides a kit for diagnosing vascular stenosis, comprising the composition for diagnosing vascular stenosis. Preferably, the kit may be an Enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

Also, preferably, the kit for diagnosing vascular stenosis may further include one or more other component compositions, solutions, or devices suitable for the analysis method.

Also preferably, it may be a diagnostic kit characterized by including the necessary elements necessary to perform the ELISA. ELISA kits include antibodies specific for the protein. The antibody is an antibody having high specificity and affinity for each marker protein and having little cross-reactivity to other proteins. The antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit can also include antibodies specific for the control protein. Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.

In addition, it may be a rapid kit, characterized in that it contains the necessary elements necessary to perform a quick test that can know the analysis results within 5 minutes. Rapid kits include antibodies specific for a protein. The antibody is an antibody having high specificity and affinity for each marker protein and having little cross-reactivity to other proteins. The antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. Rapid kits may also include antibodies specific for the control protein. Other rapid kits include reagents that can detect bound antibodies, such as nitrocellulose membranes to which specific and secondary antibodies are immobilized, membranes bound to beads to which antibodies are bound, absorbent pads, and sample pads. Substances and the like.

Also preferably, it may be a MRM (Multiple reaction monitoring) kit, which is an MS / MS mode characterized by including the necessary elements necessary for performing mass spectrometry. Whereas Selected Ion Monitoring (SIM) is a method of using ions generated by collisions once in the source portion of the mass spectrometer, MRM selects a particular ion one more time from the broken ion to detect another continuously connected source of MS. It is a method of using the ions obtained from the collision after passing through it once more. More specifically, in SIM, there is a problem in that the selected ions may interfere with quantification when the selected ions are ions that are also detected in plasma. On the other hand, in case of using MRM, even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. Can be obtained. Thus, by using the MRM mode in mass spectrometry it is possible to simultaneously analyze the desired materials with better analytical sensitivity. Through the multiple reaction monitoring (MRM) analysis method, it is possible to compare the protein expression level in the non-diabetic patients with the control group and the protein expression level in individuals with vascular stenosis. By comparing the protein expression level of and the level of protein expression in individuals with vascular stenosis, it is possible to diagnose the development of vascular stenosis disease.

In another aspect, the present invention provides a method of providing information for diagnosing a vascular stenosis disease.

Preferably, the method of the present invention comprises (a) Poly (ADP-ribose) polymerase 4 (PARP4), vitamin D-binding protein (VDB) from a biological sample. ), And measuring the expression level of one or more proteins selected from the group consisting of Laminin subunit beta-1 (LAMB1); And

(b) a method of providing information for diagnosing non-diabetic vascular stenosis disease, including comparing the expression level of the protein measured in step (a) with a normal control sample.

Or preferably, the method of the present invention comprises (a) Interferon regulatory factor 7, IRF7, Dedicator of cytokinesis protein 2 (DOCK2), and hemoglobin subunit from a biological sample Measuring the expression level of one or more proteins selected from the group consisting of beta (Hemoglobin subunit beta, HBB); And

(b) comparing the expression level of the protein measured in step (a) with a normal control sample, may be a method of providing information for diagnosing diabetic vascular stenosis disease.

The term "biological sample" as used herein includes, but is not limited to, samples such as whole blood, serum, plasma, saliva, cerebrospinal fluid, or urine, which differ in protein expression levels due to the development of vascular stenosis.

The PARP4, VDB, and LAMB1 are all characterized by a change in the expression level of the protein in individuals without diabetes but with vascular stenosis compared to the normal control group (diabetic and vascular stenosis). Changes can be diagnosed as non-diabetic vascular stenosis.

The IRF7, DOCK2, and HBB are all characterized by a change in the expression level of the protein in individuals with diabetes and vascular stenosis, compared to the control (individuals with diabetes, but without stenosis) Diabetic vascular stenosis can be diagnosed.

Therefore, when the expression level of the protein of the separated sample of the subject suspected of vascular stenosis is higher or lower than the expression level of the protein of the normal control sample, it can be determined as a vascular stenosis.

Preferably the protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein. The antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the antibody is used.

As used herein, the term “antigen-antibody complex” refers to the combination of a protein antigen of interest in an biological sample with an antibody that recognizes it. The detection of the antigen-antibody complex can be detected using methods as known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.

For the purpose of the present invention, the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, radioimmunoassay, radiation Immunodiffusion, Oukteroni Immunodiffusion, Rocket Immunoelectrophoresis, Tissue Immunostaining, Complementary Assay, Two-Dimensional Electrophoresis, Liquid Chromatography-Mass Spectrometry (LCMS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), western blot, and ELISA (enzyme linked immunosorbentassay), but are not limited thereto.

In a specific embodiment of the present invention, the LC-MS / MS method was used to measure and compare protein expression levels of PARP4, VDB, LAMB1, IRF7, DOCK2, HBB, C4A, LBP, and APOC2 itself.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: Preparation of Patient's Blood Sample

Among non-diabetic patients, they are classified into low-risk group (0%), medium-risk group (0-50%), and high-risk group (more than 50%) according to the degree of vascular stenosis. The patients were classified into high risk, medium risk, and high risk patients. Blood samples were then taken from these patients.

Example  2: Removal of abundant protein from patient blood sample

Fourteen high-abundant proteins (Albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, among proteins in blood samples collected in Example 1) complement C3 (transthyretin) accounts for 95% of the total protein. Therefore, these 14 high-abundant proteins may cause significant decrease in sensitivity in the analysis of low-abundant proteins of interest, and thus the 14 proteins may be transferred to a multiple affinity removal system (MARS) column (P / N 5188-6558, Agilent). The remaining protein was used for subsequent analysis.

Specifically, using a MARF (Multiple Affinity Removal System) column (P / N 5188-6558, Agilent), the process of removing the high-abundant protein is as follows.

40 μl of the blood sample prepared in Example 1 was added 2 μl of 50x protease inhibitor (Roche) and mixed with 60 μl of Buffer A. Then, a sample mixed with the protease inhibitor and Buffer A was added to a 0.22 μm filter (Agilent, P / N 5185-5990) using a centrifuge (14400 g, 1 min, 4 ° C.) to remove large particles. . Then, MARS column was connected to HPLC and mobile phase A (Buffer A) and B (Buffer B) were sent out.

After stabilizing the column for about 30 minutes using mobile phase A, samples were separated using the mobile phase gradient under the conditions shown in Table 1 below, and flow-through (9-17 min) and elution (21-23 min). Samples of time were transferred to 2 ml tubes. The sample obtained in the flow-through was placed in a 10 K filter tube and concentrated using a centrifuge (14400 g, 30 min, 4 ° C.) to a concentration of 2 ml of the sample to 50 μl.

Example  3: protein sample Peptidation

For the analysis by mass spectrometer, the process of fragmenting a protein into peptide units using an enzyme (trypsin) should be preceded. Thus, peptide fragmentation of the protein was carried out using the following process.

150 μl of DTT solution (final concentration 6M urea, 10mM DTT, 50mM tris) was added to 50 μl of blood sample corresponding to the concentration of 60 μg from which the abundant protein was removed and reacted at 37 ° C. for 1 hour. The 200 μl reducted sample was treated with 20 μl of IAA solution (final concentration: 5.45 M urea, 50 M IAA, 50 mM tris) and then reacted (alkylated) at room temperature in the dark for 30 minutes. Afterwards, 1 ml Tris solution (50 mM pH 8) (final concentration; 1 M urea, 50 ml tris final volume 1.2 ml) was added to the sample. Thereafter, 5 μl of trypsin solution was dispensed and reacted at 37 ° C. for 12 hours.

Example  4: Peptide  Sample TMT Tandem Mass Tag 6plex  Labeling with Reagents

Using a TMT-6plex reagent commonly used for relative quantification in mass spectrometry, a total of six sample groups were reacted with TMT reagents having different masses of reporter ions (Table 2). Table 2 shows the types of reporter ions labeled by group.

50 μL of 200mM TEAB was added to the peptide-ized sample in Example 3, and vortexing and spin down were performed. Write the name of the sample in the TMT reagent tube, add 41 μL of 100% ACN to the TMT reagent, and perform vortexing and spin down (the amount of ACN contained in each kit is determined and peptide 25-100 μg 41 μL of ACN). Then, 41 μL of TMT reagent was added to a sample tube (Peptide), and then reacted at room temperature for 1 hour after vortexing and spin down. 8 μL of 5% Hydroxylamine was added to each sample tube, followed by vortexing and spin down, followed by quenching at room temperature for 15 minutes. Thereafter, 99uL of quantitative and equivalent labeled samples were collected and collected in one new e-tube.

Example  5: Peptide Fractionation LC, MS / MS and data analysis

In order to reduce the high level of complexity of the peptide sample, fractionation was performed using the unique PI value of the peptide.

First, the TMT reagent-labeled peptide was dissolved in an offgel dilution buffer. After combining IPG strip and comb to Offgel fractionators (3100 offgel fractionator, Agilent), samples dissolved in the buffer were dispensed and run at 4000 V for 24 hours. Samples in each fraction were transferred to tubes and dried, and desalting was performed to remove reagents remaining in the samples.

The obtained peptides were separated by the hydrophobicity of the peptide by using a reverse-phase column in Easy-nLC (Thermo), a UPLC system with high resolution and high reproducibility. Peptides isolated by UPLC were analyzed in real time by Q-exactive (Thermo), a mass spectrometer with high resolution and high accuracy. Raw data obtained by Q-exactive were analyzed by spectrum analysis using the Proteome Discoverer software (Thermo) SEQUEST algorithm to identify peptides and proteins. Validation of the identified protein was performed using Scaffold Q + (Proteome software), and the quantitative information obtained from TMT was extracted. Statistical analysis was performed using Isobar, one of the R packages, and proteins with significant differences in expression levels were selected between the groups. (p value <0.05)

Table 3 shows a group of 100 candidate proteins whose expression levels differed significantly by the difference in vascular stenosis in the non-diabetic / diabetic group.

Example  6: MRM  Bio through (Multiple Reaction Monitoring) Marker  Candidate Verification

The MRM transition of the primary biomarker candidate group was selected to verify the biomarker candidate. Up to three peptides were selected per protein and three transition lists per peptide were selected. The selection of MRM transition was done using skyline software (https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view), and the transition was made using the MIST transition database provided by NIST. Selected. The selection criteria are as follows.

1.6 ≤ Length ≤ 20

2.Charge state = +2 or +3

3.No M, W (Oxidation)

NTT = 2

5. NMC = 0

Neighboring KR = 0

The selected peptide transition was performed prior to MRM analysis using blood samples, and the suitability of the transition was confirmed. The conditions for confirming transition are as follows.

1. The dot-product score provided by the skyline is 0.8 or above.

2. Whether three transition peaks derived from the same MS1 co-elution at the same retention time.

3. Whether it has reproducible retention time through three iterations.

4. Whether the peak area intensity is greater than S / N> 10.

Among the 100 candidate proteins, 6 proteins corresponding to PARP4, C4A, APOC2, LBP, VDB, and LAMB1 were identified as proteins with significant differences in expression according to the presence or absence of vascular stenosis in non-diabetic patients (Fig. 1). Specifically, among the validated proteins, PARP4 decreased the expression of PARP4 protein in the medium risk group compared to the low risk group, C4A decreased the expression of C4A protein in the high risk group, and APOC2 compared to the low risk group. The expression of APOC2 protein was increased in both the high risk and high risk groups, and VDB decreased the expression of VDB protein in both the high risk and high risk groups, and LAMB1 protein was increased in both the high risk and high risk groups. The expression of LBP was decreased, and the expression of LBP was increased in the high risk group compared to the medium risk group.

In addition, among the 100 candidate proteins, 6 proteins corresponding to DOCK2, C4A, APOC2, IRF7, HBB, and LBP were identified as having significant differences in expression according to the presence or absence of vascular stenosis in diabetic patients ( See FIG. 2). Specifically, the expression of IRF7 increased the expression of IRF7 protein in both the high risk and the high risk group compared to the low risk group, and DOCK2 decreased the expression of the DOCK2 protein in both the high risk and high risk groups compared to the low risk group. The expression of APOC2 protein was decreased in both the high risk and high risk groups compared to the low risk group, HBB increased the expression of HBB protein in the high risk group compared to the low risk group, and LBP was higher in both the high risk group and the low risk group. The expression of LBP protein was increased, and the expression of C4A was increased in the high risk group compared to the medium risk group.

Table 4 shows the peptide sequences used for MRM (Multiple Reaction Monitoring), which shows the peptide sequences of proteins that can diagnose non-diabetic / diabetic vascular narrowing.

Claims (12)

Poly (ADP-ribose) polymerase 4 (PARP4), Vitamin D-binding protein (VDB) and Laminin subunit beta-1, A composition for diagnosing non-diabetic vascular stenosis disease, comprising an agent for measuring at least one protein level selected from the group consisting of LAMB1). One selected from the group consisting of Interferon regulatory factor 7, IRF7, Dedicator of cytokinesis protein 2 (DOCK2), and hemoglobin subunit beta (HBB) Comprising an agent for measuring the above protein level, diabetic vascular stenosis disease diagnostic composition. The composition for diagnosing vascular stenosis disease according to claim 1 or 2, wherein the agent for measuring the protein level comprises an antibody that specifically binds to the protein. According to claim 1 or 2, wherein the vascular stenosis disease is stroke, atherosclerosis, in-stent restenosis, myocardial infarction, or atherosclerosis, composition for diagnosing vascular stenosis disease. A kit for diagnosing a vascular stenosis disease, comprising the composition of claim 1. The kit for diagnosing vascular stenosis according to claim 5, wherein the kit is an Enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. (a) Poly (ADP-ribose) polymerase 4 (PARP4), Vitamin D-binding protein (VDB), and laminin subunit beta-1 from biological samples Measuring the expression level of one or more proteins selected from the group consisting of (Laminin subunit beta-1, LAMB1); And (b) comparing the expression level of the protein measured in step (a) with a normal control sample, the method of providing information for diagnosing non-diabetic vascular stenosis disease. (a) From a biological sample to Interferon regulatory factor 7, IRF7, Dedicator of cytokinesis protein 2 (DOCK2), and hemoglobin subunit beta (HBB) Measuring at least one protein expression level selected from the group consisting of; And (b) comparing the expression level of the protein measured in step (a) with a normal control sample, the method of providing information for diagnosing diabetic vascular stenosis disease. The method of claim 7 or 8, wherein when the expression level of the protein of the isolated sample of the subject suspected of vascular stenosis is higher or lower than the expression level of the protein of the normal control sample, it is determined that the vascular stenosis. To provide information for diagnosing vascular stenosis. The method of claim 7 or 8, wherein the vascular stenosis disease is stroke, atherosclerosis, in-stent restenosis, myocardial infarction, or atherosclerosis. . The method of claim 7 or 8, wherein the protein expression level is measured using an antibody that specifically binds to the protein. The method of claim 7 or 8, wherein the measurement or comparison of protein expression levels is performed by protein chip analysis, immunoassay, ligand binding assay, Matrix Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, radioimmunoassay, Radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC- Method for providing information for diagnosing vascular stenosis disease, characterized in that performed using a selected from the group consisting of MS / MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbentassay) .

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