阅读说明：本技术 尿液蛋白标志物在诊断颈动脉狭窄和缺血性脑卒中的用途 (Application of urine protein marker in diagnosis of carotid stenosis and cerebral arterial thrombosis ) 是由 吴建强 王威 郑月宏 于 2021-08-03 设计创作，主要内容包括：本公开涉及尿液蛋白标志物在诊断颈动脉狭窄和缺血性脑卒中的用途。具体而言,本公开涉及在颈动脉狭窄患者中利用尿液蛋白标志物诊断和早期预测缺血性脑卒中的用途,所述尿蛋白标志物包括溶酶体酸性磷酸酶、磷脂酶D3、N-乙酰氨基半乳糖-6-硫酸酯酶、Culbin、含蛋白3的NHL重复序列、胱抑素-S、溶酶体α-葡萄糖苷酶、甲酰胺酰基转移酶环脱氨酶、α-半乳糖苷酶A等。(The present disclosure relates to the use of urine protein markers for the diagnosis of carotid stenosis and ischemic stroke. In particular, the present disclosure relates to the use of urine protein markers including lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal α -glucosidase, formamide acyltransferase cyclic deaminase, α -galactosidase a, and the like, for the diagnosis and early prediction of ischemic stroke in patients with carotid stenosis.)

1. Use of an identification agent for the manufacture of a diagnostic device for carotid stenosis in a subject, said identification agent specifically identifying any one or a combination of proteins selected from the group consisting of:

lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase cyclodeaminase, α -galactosidase A, arylsulfatase F, lysosome protective protein, α -N-acetylglucosaminidase, granule protein precursor, γ -glutamyl hydrolase, β -glucuronidase, putamenin, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, serine hydroxymethyltransferase, aminoacylase-1, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, calcium binding protein 39, betaine-homocysteine S-methyltransferase, Cytoplasmic C-1-tetrahydrofolate synthase, aspartic protease A, arylsulfatase A, acid ceramidase, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, low density lipoprotein receptor, peroxidase-6, β -hexosaminidase subunit α, voltage-dependent anion selective channel protein 1, triple kinase/FMN cyclase, galactosidase, cystatin-S, immunoglobulin λ variant 4-60, immunoglobulin λ variant 3-19, adaptor adhesion molecule A, cathelicidin, smooth muscle myosin light chain kinase, tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, cystatin-SA, cysteine-rich secreted protein 3, ADAMTS-like protein 4, carboxypeptidase Z, paracytolysin, complement component C9;

preferably, lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal α -glucosidase, formamide acyltransferase ring deaminase, α -galactosidase a and arylsulfatase F;

the protein is a protein in urine;

the diagnostic device is a kit or chip.

2. Use of an identification agent for the manufacture of a diagnostic device for the early prediction of ischemic stroke in a subject, said identification agent specifically identifying any one or a combination of proteins selected from the group consisting of:

immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, folate receptor beta, carboxypeptidase N catalytic chain, vinca protein, HLA class I histocompatibility antigen B-51 alpha chain, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit zeta, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, glypican-specific phospholipase D, nicotinic acid phosphoribosyltransferase, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, protein containing thioredoxin domain 17, endoplasmic reticulopeptidase 1, protein variants of the protein, and the like, Plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras related protein Ral-A, interleukin-2 receptor beta subunit, peptidyl-prolyl cis-trans isomerase B, testosterone-1, titin, gamma-glutamyl hydrolase, golgi protein 1;

preferably, immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-associated protein Ral-A, and folate receptor beta;

wherein the subject is a carotid stenosis patient;

the protein is a protein in urine;

the diagnostic device is a kit or chip.

3. Use according to any one of claims 1 to 2, the identification agent being selected from: mass spectrometric identification of reagents, antibodies or antigen binding fragments thereof.

4. The use of claim 3, wherein the antibody is a monoclonal antibody.

5. The use of claim 3, wherein the mass spectrometry identification reagent comprises a mass spectrometry identification parameter.

6. Use according to claim 1, wherein:

indicating that the subject has or is at increased risk of having carotid stenosis when the expression level of a protein selected from any one or a combination of: lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase cyclodeaminase, α -galactosidase A, arylsulfatase F, lysosome protective protein, α -N-acetylglucosaminidase, granule protein precursor, γ -glutamyl hydrolase, β -glucuronidase, putamenin, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, serine hydroxymethyltransferase, aminoacylase-1, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, calcium binding protein 39, betaine-homocysteine S-methyltransferase, Cytoplasmic C-1-tetrahydrofolate synthase, aspartic protease A, arylsulfatase A, acid ceramidase, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, low density lipoprotein receptor, peroxidase-6, β -hexosaminidase subunit α, voltage-dependent anion selective channel protein 1, triskinase/FMN cyclase, galactosidase; preferably, lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase ring deaminase, α -galactosidase a and arylsulfatase F;

an increased expression level of a protein selected from any one or a combination of: cystatin-S, immunoglobulin λ variant 4-60, immunoglobulin λ variant 3-19, connexol molecule a, cathelicidin antimicrobial peptide, smooth muscle myosin light chain kinase, tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, cystatin-SA, cysteine-rich secretory protein 3, ADAMTS-like protein 4, carboxypeptidase Z, paracytolysin, complement component C9; preferably, cystatin-S;

the control was an individual who did not have carotid stenosis and did not have stroke.

7. Use according to claim 2, wherein:

an increased risk of developing stroke in the subject is indicated by an increased expression level of a protein selected from any one or a combination of:

immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, folate receptor beta, carboxypeptidase N catalytic chain, vinculin, HLA class I histocompatibility antigen B-51 alpha chain, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit ζ, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, glypican-specific phospholipase D, nicotinic acid phosphoribosyltransferase, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, protein 17 containing thioredoxin domain, endoplasmic reticulum aminopeptidase 1; preferably, immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, and folate receptor beta;

an increased risk of developing stroke in the subject is indicated when the expression level of a protein selected from any one or a combination of: plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras related protein Ral-A, interleukin-2 receptor beta subunit, peptidyl-prolyl cis-trans isomerase B, testosterone-1, titin, gamma-glutamyl hydrolase, golgi protein 1; preferably, plasma serine protease inhibitors, lipoprotein lipase, β -glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras-related protein Ral-a;

the control was asymptomatic carotid stenosis patients.

8. A kit or chip for diagnosing carotid stenosis comprising an identification reagent that specifically identifies any protein or combination thereof selected from the group consisting of:

lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase cyclodeaminase, α -galactosidase A, arylsulfatase F, lysosome protective protein, α -N-acetylglucosaminidase, granule protein precursor, γ -glutamyl hydrolase, β -glucuronidase, putamenin, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, serine hydroxymethyltransferase, aminoacylase-1, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, calcium binding protein 39, betaine-homocysteine S-methyltransferase, Cytoplasmic C-1-tetrahydrofolate synthase, aspartic protease A, arylsulfatase A, acid ceramidase, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, low density lipoprotein receptor, peroxidase-6, β -hexosaminidase subunit α, voltage-dependent anion selective channel protein 1, triple kinase/FMN cyclase, galactosidase, cystatin-S, immunoglobulin λ variant 4-60, immunoglobulin λ variant 3-19, adaptor adhesion molecule A, cathelicidin, smooth muscle myosin light chain kinase, tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, cystatin-SA, cysteine-rich secreted protein 3, ADAMTS-like protein 4, carboxypeptidase Z, paracytolysin, complement component C9; preferably, lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal α -glucosidase, formamide acyltransferase ring deaminase, α -galactosidase a and arylsulfatase F;

the identifying agent is selected from: mass spectrometric identification of reagents, antibodies or antigen binding fragments thereof;

preferably, the mass spectrometric identification reagent comprises mass spectrometric identification parameters;

preferably, the antibody is a monoclonal antibody.

9. A kit or chip for early prediction of ischemic stroke comprising an identifying agent: the identification agent specifically identifies any one or a combination of proteins selected from the group consisting of:

immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, folate receptor beta, carboxypeptidase N catalytic chain, vinca protein, HLA class I histocompatibility antigen B-51 alpha chain, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit zeta, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, glypican-specific phospholipase D, nicotinic acid phosphoribosyltransferase, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, protein containing thioredoxin domain 17, endoplasmic reticulopeptidase 1, protein variants of the protein, and the like, Plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras related protein Ral-A, interleukin-2 receptor beta subunit, peptidyl-prolyl cis-trans isomerase B, testosterone-1, titin, gamma-glutamyl hydrolase, golgi protein 1;

preferably, immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-associated protein Ral-A, and folate receptor beta;

the identification reagent is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof;

preferably, the mass spectrometric identification reagent comprises mass spectrometric identification parameters;

preferably, the antibody is a monoclonal antibody.

Technical Field

The present disclosure relates to clinical medicine; in particular to a urine protein marker related to human carotid artery stenosis. In particular, the disclosure relates to a human carotid artery stenosis diagnosis and/or early prediction ischemic stroke related urine protein marker obtained by using a carotid artery stenosis patient urine sample and a mass spectrometry proteomics technology, and application thereof.

Background

Carotid Artery Stenosis (CAS) is characterized by the formation of atherosclerotic plaques in the carotid artery, accounting for approximately 20-30% of all strokes. Stroke is the second leading cause of death and adult disability worldwide, and thus prevention of stroke is of great social value.

The progression of CAS is insidious and patients may continue asymptomatic until stroke or transient ischemic attack symptoms occur. Therefore, early diagnosis of CAS is crucial to the management of CAS. CAS can be clinically classified as symptomatic and asymptomatic CAS. With symptomatic CAS patients having a significantly increased risk of stroke and the clinical management of symptomatic and asymptomatic CAS patients also differing. Characterizing symptomatic CAS facilitates early prediction, risk stratification, and early intervention of stroke.

In the past decade, mass spectrometry-based proteomic analysis has revolutionized the discovery of disease biomarkers by detecting thousands of proteins from various sample sources. The urine has the advantages of no wound, simple collection, stable state, no inhibition of high-abundance protein and the like, so the urine becomes an extremely attractive biomarker source in clinical proteomics research.

Thus, there remains a need in the art for protein markers that can distinguish CAS patients from controls, and that can distinguish symptomatic CAS patients from asymptomatic CAS patients, potentially providing a non-invasive and effective method for CAS diagnosis and risk stratification.

Disclosure of Invention

In view of the above-mentioned need in the art, according to some embodiments of the present disclosure, there is provided a use of an identifying agent selected from any one of the following proteins in the manufacture of a diagnostic device for carotid stenosis:

lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal alpha-glucosidase, formamide acyltransferase cyclic deaminase, alpha-galactosidase A, arylsulfatase F, lysosomal protective protein, immunoglobulin lambda variant 4-60, alpha-N-acetylglucosaminidase, granule protein precursor, immunoglobulin lambda variant 3-19, adaptor adhesion molecule A, gamma-glutamyl hydrolase, beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, gamma-glutamyl hydrolase, alpha-galactosidase-alpha-beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, and alpha-beta-glucosidase, Tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, aminoacylase-1, cystatin-SA, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, cytoplasmic C-1-tetrahydrofolate synthase, aspartic proteinase A, arylsulfonase A, acid ceramidase, cysteine-rich secretory protein 3, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, ADAMTS-like protein 4, low density lipoprotein receptor, peroxidase-6, carboxypeptidase Z, beta-hexosaminidase subunit alpha, paracytoin, thrombospondin, and the like, Voltage-dependent anion-selective channel protein 1, triple kinase/FMN cyclase, complement component C9, galactosidase, or a combination thereof; preferably, the lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal α -glucosidase, formamide acyltransferase ring deaminase, α -galactosidase a and arylsulfatase F.

In particular embodiments, an increased level of expression of a protein selected from the group consisting of: cystatin-S, immunoglobulin lambda variant 4-60, immunoglobulin lambda variant 3-19, connexol a, cathelicidin, smooth muscle myosin light chain kinase, tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, cystatin-SA, cysteine-rich secreted protein 3, ADAMTS-like protein 4, carboxypeptidase Z, paracytolysin, complement component C9, or a combination thereof. Otherwise, the risk is reduced.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of: lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase cyclodeaminase, α -galactosidase A, arylsulfatase F, lysosome protective protein, α -N-acetylglucosaminidase, granule protein precursor, γ -glutamyl hydrolase, β -glucuronidase, putamenin, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, serine hydroxymethyltransferase, aminoacylase-1, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, calcium binding protein 39, betaine-homocysteine S-methyltransferase, Cytoplasmic C-1-tetrahydrofolate synthase, aspartic protease A, arylsulfatase A, acid ceramidase, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, low density lipoprotein receptor, peroxidase-6, β -hexosaminidase subunit α, voltage-dependent anion selective channel protein 1, triple kinase/FMN cyclase, galactosidase, or a combination thereof. Otherwise, the risk is reduced.

In embodiments relating to the diagnosis of carotid stenosis, a control refers to an individual who has not suffered from carotid stenosis (and preferably also has not suffered from stroke). The control need not be a clinically healthy individual, preferably a healthy individual.

In another embodiment, there is provided the use of an agent for identifying a protein selected from the group consisting of: immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3 '-phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-related protein Ral-A, folate receptor beta, interleukin-2 receptor beta subunit, carboxypeptidase N catalytic chain, focal adhesion protein, HLA class I histocompatibility antigen B-51 alpha chain, prolyl cis-trans isomerase B, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit ζ, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, beta-glucuronidase, 2', 3 '-cyclic nucleotide 3' -phosphodiesterase, and the like, Glypican-specific phospholipase D, testosterone-1, nicotinic acid phosphoribosyltransferase, titin, gamma-glutamyl hydrolase, golgi protein 1, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, thioredoxin domain-containing protein 17, endoplasmic reticulum aminopeptidase 1, or a combination thereof; preferably, the immunoglobulin lambda variant 3-9, plasma serine protease inhibitors, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-associated protein Ral-A and folate receptor beta.

In a specific embodiment, the early prediction of ischemic stroke is in a patient with carotid artery stenosis (CAS patient).

In a specific embodiment, samples are taken from CAS patients, and the levels of protein in the urine of symptomatic CAS patients and asymptomatic CAS patients are compared to predict ischemic stroke based on changes in protein markers in the urine of the patients.

Symptomatic CAS is defined as sudden focal nervous system symptoms corresponding to carotid distribution, including but not limited to: focal neurological dysfunction, transient monocular blindness, transient ischemic attacks, small (non-disabling) ischemic stroke. This definition was based on the occurrence of carotid symptoms within the past 6 months.

Asymptomatic CAS is defined as the presence of carotid stenosis in individuals who have no recent history of ischemic stroke or transient cerebral ischemia in the ipsilateral carotid artery perfusion area.

In particular embodiments, an increased expression level of a protein selected from the group consisting of: immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, folate receptor beta, carboxypeptidase N catalytic chain, vinca protein, HLA class I histocompatibility antigen B-51 alpha chain, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit zeta, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, glypican-specific phospholipase D, nicotinic acid phosphoribosyltransferase, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, protein containing thioredoxin domain 17, endoplasmic reticulopeptidase 1, protein variants of the protein, and the like, Or a combination thereof. Otherwise, the risk is reduced.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of: plasma serine protease inhibitors, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras-related protein Ral-a, interleukin-2 receptor beta subunit, peptidyl-prolyl cis-trans isomerase B, testosterone-1, titin, gamma-glutamyl hydrolase, golgi protein 1, or combinations thereof. Otherwise, the risk is reduced.

The identification agents of the present application are capable of identifying, or binding to, or searching for, or monitoring, or targeting such tagged peptides (e.g., peptides comprised by the proteins listed in table 1 or table 2). In other embodiments, the identification agents of the present application are capable of recognizing, or binding, or searching for, or monitoring, or targeting epitopes of proteins (e.g., epitopes in proteins listed in table 1 or table 2, linear or nonlinear epitopes are suitable).

In some embodiments, an identification reagent suitable for use in the present disclosure is a mass spectrometry identification reagent, an antibody, or an antigen binding fragment thereof.

In a specific embodiment, the antibody is a monoclonal antibody. The species source of the monoclonal antibody is not limited by the present disclosure, and any antibody capable of binding to the above-described protein can be used.

In particular embodiments, antigen-binding fragments include, but are not limited to: fab, Fab ', (Fab')₂Fv, ScFv, bispecific antibody, trispecific antibody, tetraspecific antibodyHetero-antibodies, bis-scFv, mimi antibodies. Any antibody fragment that retains antigen binding activity is suitable for use in the present disclosure.

In one embodiment, when mass spectrometric identification reagents are used, the identification reagents are widely understood and cannot be interpreted as merely chemical or biological reagents for the presence of the entity. The term also encompasses mass spectrometric identification parameters. As an example, the whole full scan range of the mass spectrum is divided into several windows, and all ions in each window are selected, fragmented and detected at high speed and cyclically, so that all fragment information of all ions in the sample is obtained without omission or difference. The acquisition method is like blanket bombing, and all targets are hit without omission; and comparing the mass-to-charge ratio information of the fragments in a database to identify the identity and the quantification of the protein. It should be understood that although a specific identification method is used in the specific example, the technical effect of the present application is not achieved depending on the specific identification method (e.g., mass spectrometry procedure, mass spectrometer model, parameters set in the mass spectrometry method, specific peptide sequence identified in the mass spectrometry, chromatography column model; antibody supplier, specific epitope targeted by antibody, antibody typing, immunological procedure and parameters), because the core of the technical solution of the present application is to find the relationship between the amount of the aforementioned protein present in urine and the disease, and thus any means capable of determining the protein content is available.

The tag peptide (also referred to as specific peptide herein) refers to a peptide fragment capable of representing a protein, and is characterized by the presence and specificity only in the amino acid sequence of the protein.

It is to be understood that although the specific examples identify and quantify proteins based on a particular sequence, this does not mean that peptide fragments at other positions in the proteins listed in table 1 or table 2 cannot be used, as long as such fragments are capable of distinguishing different proteins from each other, and are suitable for use herein. The position or length of the fragments can be determined by the skilled person in accordance with conventional techniques in combination with the operational requirements of the identification method used, given the teaching of the present application.

In a specific embodiment, the expression level is a protein level.

In a specific embodiment, the expression level is the level of protein expression in a urine sample from the subject.

In specific embodiments, the subject is a human.

In an aspect of the present disclosure, the patient with carotid artery stenosis is a patient with extracranial carotid artery stenosis.

In particular embodiments, the protein markers according to the present disclosure can be used for diagnosing and/or stratifying carotid stenosis.

According to other embodiments, there is also provided a kit or chip for diagnosing carotid stenosis, comprising or consisting of an identifying agent for a protein selected from the group consisting of: lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal alpha-glucosidase, formamide acyltransferase cyclic deaminase, alpha-galactosidase A, arylsulfatase F, lysosomal protective protein, immunoglobulin lambda variant 4-60, alpha-N-acetylglucosaminidase, granule protein precursor, immunoglobulin lambda variant 3-19, adaptor adhesion molecule A, gamma-glutamyl hydrolase, beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, gamma-glutamyl hydrolase, alpha-galactosidase-alpha-beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, and alpha-beta-glucosidase, Tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, aminoacylase-1, cystatin-SA, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, cytoplasmic C-1-tetrahydrofolate synthase, aspartic proteinase A, arylsulfonase A, acid ceramidase, cysteine-rich secretory protein 3, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, ADAMTS-like protein 4, low density lipoprotein receptor, peroxidase-6, carboxypeptidase Z, beta-hexosaminidase subunit alpha, paracytoin, thrombospondin, and the like, Voltage-dependent anion-selective channel protein 1, triple kinase/FMN cyclase, complement component C9, galactosidase, or a combination thereof.

In other specific embodiments, a kit or chip for early prediction of ischemic stroke is provided, which comprises or consists of an identifying agent for a protein selected from the group consisting of: immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3 '-phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-related protein Ral-A, folate receptor beta, interleukin-2 receptor beta subunit, carboxypeptidase N catalytic chain, focal adhesion protein, HLA class I histocompatibility antigen B-51 alpha chain, prolyl cis-trans isomerase B, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit ζ, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, beta-glucuronidase, 2', 3 '-cyclic nucleotide 3' -phosphodiesterase, and the like, Glypican-specific phospholipase D, testosterone-1, nicotinic acid phosphoribosyltransferase, titin, gamma-glutamyl hydrolase, golgi protein 1, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, thioredoxin domain-containing protein 17, endoplasmic reticulum aminopeptidase 1, or a combination thereof.

In some embodiments, the kit comprises an agent for identifying the above-described protein.

In other embodiments, the chip has immobilized thereon an identifying agent for the protein.

In some embodiments, the identification agent is an antibody or antigen-binding fragment thereof.

According to some embodiments, there is provided a method for diagnosing whether a subject has carotid stenosis, comprising the steps of:

1) obtaining a urine sample from the subject or a control,

2) optionally, separating the protein from the urine sample,

3) determining the expression level of a protein selected from the group consisting of:

lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal alpha-glucosidase, formamide acyltransferase cyclic deaminase, alpha-galactosidase A, arylsulfatase F, lysosomal protective protein, immunoglobulin lambda variant 4-60, alpha-N-acetylglucosaminidase, granule protein precursor, immunoglobulin lambda variant 3-19, adaptor adhesion molecule A, gamma-glutamyl hydrolase, beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, gamma-glutamyl hydrolase, alpha-galactosidase-alpha-beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, and alpha-beta-glucosidase, Tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, aminoacylase-1, cystatin-SA, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, cytoplasmic C-1-tetrahydrofolate synthase, aspartic proteinase A, arylsulfonase A, acid ceramidase, cysteine-rich secretory protein 3, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, ADAMTS-like protein 4, low density lipoprotein receptor, peroxidase-6, carboxypeptidase Z, beta-hexosaminidase subunit alpha, paracytoin, thrombospondin, and the like, Voltage-dependent anion-selective channel protein 1, triple kinase/FMN cyclase, complement component C9, galactosidase, or a combination thereof;

4) and the protein expression level in the sample obtained from the control.

In specific embodiments, the expression level is determined using mass spectrometry, ELISA, or Western methods.

When the protein and its expression level are determined by mass spectrometry, a protease digestion step may also be included after the step of obtaining a urine sample. In a specific embodiment, the protein in the urine sample is fragmented with a protease.

According to some embodiments, there is provided a method for diagnosing a subject with carotid stenosis, comprising the steps of:

1) urine samples were obtained from the subject and from the control,

2) determining the expression level of a protein selected from the group consisting of: lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, cystatin-S, lysosomal alpha-glucosidase, formamide acyltransferase cyclic deaminase, alpha-galactosidase A, arylsulfatase F, lysosomal protective protein, immunoglobulin lambda variant 4-60, alpha-N-acetylglucosaminidase, granule protein precursor, immunoglobulin lambda variant 3-19, adaptor adhesion molecule A, gamma-glutamyl hydrolase, beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, gamma-glutamyl hydrolase, alpha-galactosidase-alpha-beta-glucuronidase, cathelicidin, slow protein, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, cytosolic serine hydroxymethyltransferase, smooth muscle myosin light chain kinase, and alpha-beta-glucosidase, Tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, aminoacylase-1, cystatin-SA, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, cytoplasmic C-1-tetrahydrofolate synthase, aspartic proteinase A, arylsulfonase A, acid ceramidase, cysteine-rich secretory protein 3, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, ADAMTS-like protein 4, low density lipoprotein receptor, peroxidase-6, carboxypeptidase Z, beta-hexosaminidase subunit alpha, paracytoin, thrombospondin, and the like, Voltage-dependent anion-selective channel protein 1, triple kinase/FMN cyclase, complement component C9, galactosidase, or a combination thereof;

3) comparing the expression level of the protein in the subject to the expression level of the protein in a control;

4) determining whether the subject has, or is assessed at risk of having, a carotid stenosis.

In particular embodiments, an increased level of expression of a protein selected from the group consisting of: cystatin-S, immunoglobulin lambda variant 4-60, immunoglobulin lambda variant 3-19, connexol a, cathelicidin, smooth muscle myosin light chain kinase, tumor necrosis factor receptor superfamily member 12A, cytoplasmic dynein 1 heavy chain 1, cystatin-SA, cysteine-rich secreted protein 3, ADAMTS-like protein 4, carboxypeptidase Z, paracytolysin, complement component C9, or a combination thereof.

In particular embodiments, a decreased level of expression of a protein selected from the group consisting of: lysosomal acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repeat containing protein 3, lysosomal α -glucosidase, formamide acyltransferase cyclodeaminase, α -galactosidase A, arylsulfatase F, lysosome protective protein, α -N-acetylglucosaminidase, granule protein precursor, γ -glutamyl hydrolase, β -glucuronidase, putamenin, peroxidase-1, Xaa preaminopeptidase 2, lysosome-related membrane glycoprotein 2, serine hydroxymethyltransferase, aminoacylase-1, calbindin 39, betaine-homocysteine S-methyltransferase 1, cytosolic 10-formyltetrahydrofolate dehydrogenase, S-methylmethionine-homocysteine S-methyltransferase BHMT2, calcium binding protein 39, betaine-homocysteine S-methyltransferase, Cytoplasmic C-1-tetrahydrofolate synthase, aspartic protease A, arylsulfatase A, acid ceramidase, cytoplasmic glycerol-3-phosphate dehydrogenase [ NAD (+) ], FAM151A protein, low density lipoprotein receptor, peroxidase-6, β -hexosaminidase subunit α, voltage-dependent anion selective channel protein 1, triple kinase/FMN cyclase, galactosidase, or a combination thereof.

According to some embodiments, there is provided a method for early prediction of ischemic stroke comprising the steps of:

1) obtaining a urine sample from a subject or control (preferably a patient with asymptomatic carotid stenosis);

2) determining the expression level of a protein selected from the group consisting of: immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3 '-phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-related protein Ral-A, folate receptor beta, interleukin-2 receptor beta subunit, carboxypeptidase N catalytic chain, focal adhesion protein, HLA class I histocompatibility antigen B-51 alpha chain, prolyl cis-trans isomerase B, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit ζ, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, beta-glucuronidase, 2', 3 '-cyclic nucleotide 3' -phosphodiesterase, and the like, Glypican-specific phospholipase D, testosterone-1, nicotinic acid phosphoribosyltransferase, titin, gamma-glutamyl hydrolase, golgi protein 1, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, thioredoxin domain-containing protein 17, endoplasmic reticulum aminopeptidase 1, or a combination thereof;

3) comparing the expression level of the protein in the subject to a control;

4) determining the risk of the subject developing ischemic stroke.

In particular embodiments, an increased expression level of a protein selected from the group consisting of: immunoglobulin lambda variant 3-9, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, folate receptor beta, carboxypeptidase N catalytic chain, vinculin, HLA class I histocompatibility antigen B-51 alpha chain, calmodulin-like protein 3, serine/threonine protein phosphatase PP1 gamma catalytic subunit, T-complex protein 1 subunit ζ, mitochondrial malate dehydrogenase, stanniocalcin-1, biliverdin reductase A, interleukin-13 receptor subunit alpha-1, glypican-specific phospholipase D, nicotinic acid phosphoribosyltransferase, phosphotriesterase-related protein, selectin 18, synaptophysin VAT-1 homolog, thioredoxin domain containing protein 17, endoplasmic reticulum aminopeptidase 1, or a combination thereof.

In particular embodiments, the reduced expression level of a protein selected from the group consisting of: plasma serine protease inhibitors, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, Ras-related protein Ral-a, interleukin-2 receptor beta subunit, peptidyl-prolyl cis-trans isomerase B, testosterone-1, titin, gamma-glutamyl hydrolase, golgi protein 1, or combinations thereof.

Drawings

Fig. 1A and 1B: CT examinations were performed on the neck of CAS patients and controls.

Detailed Description

The disclosure will be further illustrated by the following non-limiting examples. It will be apparent to those skilled in the art that many modifications can be made to the disclosure without departing from the spirit thereof, and such modifications are intended to be within the scope of the disclosure. The experimental materials used are all available from commercial companies, unless otherwise specified.

Example urine Collection and analysis of patients with carotid stenosis

1. Obtaining a morning urine sample from a patient with carotid stenosis; urine protein expression was compared to controls, and asymptomatic patients with carotid stenosis.

The control was a subject without carotid stenosis.

Criteria for the determination of asymptomatic carotid stenosis: see Moresol P et al, cardiac tent Versus Endarternecty for asymmetric Carotid aromatic Stenosis, A Systematic Review and Meta-analysis, Stroke 2017; 48:2150-2157.

Criteria for the determination of symptomatic carotid stenosis: see Brott, T.G. et al Long-Term Results of bearing vertuss Enterprise for vehicle-arm Stenosis.New England and Journal of Medicine 2016; 374(11),1021-1031.

2. Materials and reagents

1) The instrument comprises the following steps:

the Orbitrap Q active HF high resolution mass spectrometer was purchased from Thermo Fisher; HPLC, EASY-nLC 1200 was purchased from Thermo Fisher.

2) Reagent:

chromatographic grade acetonitrile, formic acid and methanol are produced by Waters corporation; acetylammonium Iodide (IAA), ammonium bicarbonate, Dithiothreitol (DTT) were purchased from Sigma; mass Spectroscopy grade pancreatin was purchased from Promega.

3. Experimental methods

1) Sample collection procedure: urine was collected from carotid stenosis patients and controls.

2) Extracting urine protein of a patient: centrifuging 5000g of patient urine for 30min, collecting supernatant, mixing the urine with acetone at a volume ratio of 1:4, and precipitating urine protein at-20 ℃ overnight. Centrifuging at 12000g for 30min at 4 deg.C, discarding supernatant, naturally air drying precipitate, and adding lysis solution for redissolving.

3) Preparation of proteome sample: urine proteins are cleaved on the membrane using the FAST method, see Wisniwski JR et al, Universal sample preparation method for protein analysis, Nature methods 2009; 6:359-62.

4) Mass spectrum identification: the digested peptide fragment was subjected to tandem mass spectrometry using an Orbitrap Q active HF high resolution mass spectrometer. Setting parameters: modify the mass spectrum variable window, scan time 60min, collision energy: 28%; the primary scanning resolution is set to 60000, and the secondary resolution is 30000; the maximum injection time of the parent ions is 80 ms; parent ion scan range: 300-1300 m/z; sub-ion scan range: starting from 200 m/z; setting 40 scanning windows; the window size is determined according to the number of primary parent ions and is evenly distributed.

5) Proteome quantification: the obtained mass spectrum data is retrieved by a Proteome scanner software (version 2.1), and a result file is imported into a Spectronaut software to establish a spectrogram library. The mass spectrum raw data of the formal sample is introduced into a Spectronaut software and matched with the data in a spectrogram library, and the q value is less than 1.0%, and the retention time is corrected by iRT reagent. And screening the proteins with more than 2 specific polypeptides for subsequent proteome quantification. And screening out the protein with the protein expression quantity change fold of more than 1.5 times and the p value of less than 0.05 as the differential protein.

Test example

Test example 1 carotid CT (computed tomography) examination

The subject was examined by neck CT to determine whether carotid stenosis had occurred. The examination results were as follows:

CT imaging changes:

CT examination of the neck of CAS patients (FIG. 1A) and controls (FIG. 1B) revealed a significant narrowing of the CAS patients carotid artery.

2. Protein identification results:

urine samples were collected from 35 patients with confirmed carotid stenosis, 12 patients with symptomatic carotid stenosis and 23 patients with asymptomatic carotid stenosis, and 1394 proteins were identified in total at a level where at least 2 specific polypeptides and proteins identified FDR < 1%.

1) And comparing with a control sample, screening out the differential proteins with the change multiple of more than 1.5 times and the t test p of an independent sample of less than 0.05, and selecting the first 50 differential proteins with smaller p values as diagnostic markers.

Table 1 shows that 50 different proteins were co-screened for carotid stenosis, 14 proteins were elevated and 36 proteins were reduced.

TABLE 1 carotid stenosis patient and control differential urine protein markers

2) Compared with samples of asymptomatic carotid stenosis, symptomatic carotid stenosis was screened for differential proteins with fold change of more than 1.5 fold and independent samples t-test p < 0.05. Table 2 shows that 33, 21 proteins were elevated and 11 proteins were reduced from the co-screen.

TABLE 2 differential urine protein markers for symptomatic and asymptomatic carotid stenosis patients

Commercially available antibodies were purchased (or laboratory self-prepared specific antibodies) against the proteins in table 1. Urine samples of CAS patients and subjects were collected and the resulting antibodies were used to test the protein expression levels in the samples using a blind method. Expression levels of the table 1 proteins (used alone or in combination) were able to distinguish CAS patients from subjects by statistical analysis (p ═ 0.05) (data not shown). In particular, the sensitivity and specificity of the detection method can meet the clinical requirements when the combination of the lysosome acid phosphatase, phospholipase D3, N-acetylgalactosamine-6-sulfatase, Culbin, NHL repetitive sequence containing protein 3, cystatin-S, lysosome alpha-glucosidase, formamide acyltransferase cyclic deaminase, alpha-galactosidase A and arylsulfatase F is used in Table 1 (other markers can be selected optionally, and the sensitivity and specificity can be increased by adding the markers).

For the proteins in table 2, commercially available antibodies were purchased (or specific antibodies were prepared by the laboratory). Urine samples of symptomatic and asymptomatic carotid stenosis patients were collected and the resulting antibodies were used to test the protein expression levels in the samples using a blind method. Statistical analysis (p ═ 0.05), expression levels of the proteins of table 2 (used alone or in combination) allowed discrimination between symptomatic and asymptomatic patients with carotid stenosis, and prediction of stroke risk (data not shown). Specifically, the immunoglobulin lambda variant 3-9, plasma serine protease inhibitor, lipoprotein lipase, beta-glucuronidase, 2', 3' -cyclic nucleotide 3' -phosphodiesterase, immunoglobulin lambda-1 light chain, HLA class I histocompatibility antigen Cw-7 alpha chain, Ras-related protein Ral-A and folate receptor beta in Table 2 are combined for use (other markers can be optional, and the sensitivity and specificity can be increased by adding the markers), and the sensitivity and specificity of the detection method meet the clinical requirements.