CN111273037B - Urine protein marker for diagnosing Parkinson's disease - Google Patents

Abstract

The invention relates to a urine protein marker for diagnosing Parkinson's disease. Specifically, the invention relates to application of a reagent for detecting the protein content in urine of a subject in preparing a medicament for diagnosing Parkinson's disease of the subject, wherein the reagent for detecting the protein content in the urine of the subject is a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof.

Description

Urine protein marker for diagnosing Parkinson's disease

Technical Field

The present invention relates to the technical field of clinical diagnosis. In particular, the present invention relates to urine protein markers for the diagnosis of parkinson's disease. In particular to a urine protein marker related to human Parkinson disease and obtained by using a mass spectrometry proteomics technology and application thereof.

Background

Parkinson's disease is a neurodegenerative disease characterized by progressive movement and non-movement disorders, see Deng, H.and L.Yuan, Genetic variants and animal models in SNCA and Parkinson disease. The main pathological characteristics are progressive loss of dopaminergic neurons in the compact part of the substantia nigra of the midbrain, and eosinophilic inclusion bodies with alpha synuclein (alpha-synuclein) as a main component, namely lewy bodies, appear in the residual dopaminergic neurons. Parkinson's disease seriously affects the quality of life of patients and their families, creating a huge socio-economic burden, see Yang, y.x., n.w.wood, and d.s.latchman, Molecular basis of Parkinson's disease. neuroreport,2009.20(2): p.150-156; and Delenclos, M. et al, Biomarkers in Parkinson' S disease: Advances and strategies, Parkinson & Related Disorders,2016.22: p.S. 106-S110.

The early diagnosis of the Parkinson's disease has great significance for improving the life quality of patients and delaying the development of diseases. At present, no effective means for curing the Parkinson's disease exists, the progress of the Parkinson's disease can be delayed only through modes of medicament treatment, surgical treatment and the like, the medicament treatment is mainly used, but the curative effect is weakened and some side effects are easy to occur after the medicament is used for a long time. While the clinical diagnosis of Parkinson's disease is currently mainly based on motor characteristics, about 60-70% of neurons have been lost when motor characteristics of Parkinson's disease are manifested, see Mehta, S.H. and C.H. Adler, Advances in Biomarker Research in Parkinson's disease Current Neurology and Neuroscience Reports,2015.16(1): p.7. Because the early symptoms of the Parkinson's disease are slight and are generally considered as the result of aging, the early diagnosis of the Parkinson's disease is very difficult at present, a certain misdiagnosis rate exists, and most patients have obvious behavior disorder and accumulated pathological changes when the diagnosis is confirmed. Therefore, in order to provide better clinical treatment for parkinson patients, and to help researchers find new therapeutic approaches, there is an urgent need to find sensitive early biomarkers.

Biomarkers are a class of indicators that objectively reflect normal physiological, pathological processes, see Strimbu, k.and j.a.tavel, white are biornarkers curr Opin HIV AIDS,2010.5(6): p.463-6. The most studied biomarkers are still present in blood and cerebrospinal fluid. Cerebrospinal fluid is considered to be an ideal choice for finding biomarkers for neurodegenerative diseases because there is no barrier between it and the brain, but is more stable and more difficult to obtain than blood due to the presence of the blood brain barrier. During the disease process, early minor changes caused by the disease are likely to be eliminated by blood and cerebrospinal fluid before reaching the critical point of decompensation due to homeostasis regulation, and Urine has no homeostatic mechanism, so that Urine can accommodate more changes and is more likely to find sensitive early markers than more stable cerebrospinal fluid and blood, see Gao, y., urea-an unused gold mine for biomaker discovery science Life Sciences,2013.56(12): p.1145-1146; and Adachi, J et al, The human urinary protein compositions more than 1500proteins, including a large protein of membrane proteins biology 2006.7(9): p.R80-R80. It has been shown that Urine can sensitively reflect the changes in the development of neurodegenerative diseases, for example, in Transgenic mice with Alzheimer's Disease, 29 proteins with significant differences in Urine were identified in the absence of pathological β -Amyloid plaques, see Zhang, F. et al, Early cancer urea biomaterials for Detecting Alzheimer's Disease Amyloid-beta plate Deposition in an APP (swe)/PSEN1dE9Transgenic Mouse model J Alzheimer's Dis 2018.66(2): p.613-637. There are 18 significantly varying metabolites in the urine of Parkinson's disease patients that are associated with the disease stage, see Luan, H. et al, Comprehensive urinary metabolic profiling and identification of potential biochemical markers for idiophatic Parkinson's disease. Sci Rep,2015.5: p.13888.

The animal model can not only prevent urine from being influenced by various factors such as age, diet and Physiological conditions, see Wu, J.and Y.Gao, Physiological conditions can be reflected in human urine proteins and metabolism, expert Review of the properties, 2015.12(6): p.623-636, but also monitor early disease course. Overexpression of the human Alpha Synuclein A53T mutation can summarize two typical neuropathological abnormalities of Parkinson's Disease, namely progressive loss of dopaminergic neurons in the nigrostriatal and formation of Alpha Synuclein inclusion bodies, see Spillentini, M.G. et al, Alpha-Synuclein in Lewy bodies. Nature,1997.388(6645): p.839-40, widely used in Related studies in Parkinson's Disease, see Chen, X. et al, Long metallic interactions Profiling of Parkinson's Disease-Related Alpha-Synuclein A53T therapeutic Mice. PLoS One,2015.10(8): p.e 0136612; zhang, s., q.xiao, and w.le, alfactory dynamic and neutron disturbance in an alcoholic bulb of a generic chemical expressing human a53T mutant alpha-synuclein.plos One,2015.10(3): p.e 0119928; and Rothman, S.M. et al, Metabolic antigens and hyproleptimia in alpha-synuclein A53T mutant mice neurobiol Aging,2014.35(5): p.1153-61.

The influence of the occurrence and development of the Parkinson's disease on the urine proteome is explored, and a meaningful clinical index can be provided for the early diagnosis of the Parkinson's disease.

Disclosure of Invention

The inventor utilizes an alpha synuclein A53T transgenic mouse model to collect urine samples of mice for 2 months, 4 months, 6 months, 8 months and 10 months respectively, and performs quantitative analysis by liquid chromatography-tandem high resolution mass spectrometry (LC-MS/MS) based on a Data Independent Acquisition (DIA) technology. A total of 17 human homologous difference proteins were identified compared to 2 month urine samples, of which 9 proteins were reported to be associated with parkinson's disease, with the proteinogen-2, splicing factor 3A subunit 1, isopentenyl diphosphate isomerase delta-isomerase 1 changing continuously over 6, 8,10 months. Through the experiments and analysis, the urine proteome can reflect the development change of the alpha synuclein transgenic mouse and the early biomarker information, and provide clues for the clinical early diagnosis of the Parkinson's disease.

In one aspect, the present invention provides the use of an agent for detecting the amount of protein in urine of a subject, wherein the protein in the urine is selected from one or more of the following, or a combination thereof, in the manufacture of a medicament for diagnosing parkinson's disease in said subject: protein TOPAZ1, actin, elongation factor 1- α 1, thioredoxin dependent peroxidase reductase, isopentenyl diphosphate isomerase δ -isomerase 1, odour-binding protein 2a, splicing factor 3A subunit 1, PDZK1 interacting protein 1, proten-2, complement factor B, α -N-acetyl galactosidase, guanylate cyclase activator 2B, tripeptidylpeptidase 1, branched-chain amino acid aminotransferase, zinc transporter ZIP13, protein LEG1 homolog, and odour-binding protein 2 a.

Specifically, the invention provides a use of an agent for detecting the content of protein in urine of a subject in the preparation of a medicament for early diagnosis of Parkinson's disease in the subject, wherein the early diagnosis is defined as the subject having no obvious behavioral abnormality, and preferably the protein in the urine is a combination of the protein TOPAZ1 and actin.

Further, the use of the reagent for detecting the protein content in urine of a subject provided according to the present invention in the preparation of a medicament for diagnosing parkinson's disease of the subject, wherein the protein in the urine is a combination of isopentenyl diphosphate isomerase delta-isomerase 1, splicing factor 3A subunit 1 and proteogen-2; preferably, wherein the protein in the urine further comprises one or more selected from the group consisting of: protein TOPAZ1, actin, elongation factor 1- α 1, thioredoxin-dependent peroxiredoxin reductase, odorant binding protein 2a, PDZK1 interacting protein 1, complement factor B, α -N-acetylgalactosamine, guanylate cyclase activator 2B, tripeptidyl peptidase 1, branched chain amino acid aminotransferase, zinc transporter ZIP13, protein LEG1 homolog and odorant binding protein 2 a.

Further, the use provided according to the present invention, wherein the reagent for detecting the protein content in urine of a subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; preferably, wherein the reagent for detecting the protein content in the urine of the subject is a monoclonal antibody.

Still further, there is provided the use according to the present invention, wherein the subject is a mammal, preferably a human, a mouse, a rat or a companion animal.

Further, there is provided according to the invention a use wherein an increased content of actin, elongation factor 1-alpha 1, thioredoxin dependent peroxidase reductase, odour binding protein 2a, PDZK1 interacting protein 1, alpha-N-acetyl galactosidase, guanylate cyclase activator 2B, tripeptidyl peptidase 1, branched chain amino acid aminotransferase, zinc transporter ZIP13, odour binding protein 2a, and/or protein TOPAZ1, isopentenyl diphosphate isomerase delta-isomerase 1, splicing factor 3A subunit 1, proten-2, complement factor B, protein LEG1 homolog, compared to a healthy control, indicates that the subject has parkinson's disease; preferably, the healthy control is a healthy subject not suffering from parkinson's disease or other diseases.

In another aspect, the present invention provides a kit or a chip for early diagnosis of parkinson's disease, comprising a reagent for detecting the protein content in urine of a subject, wherein the protein in the urine is a combination of the following proteins: protein TOPAZ1, actin, elongation factor 1- α 1, thioredoxin dependent peroxidase reductase, isopentenyl diphosphate isomerase δ -isomerase 1, odour-binding protein 2a, splicing factor 3A subunit 1, PDZK1 interacting protein 1, proten-2, complement factor B, α -N-acetyl galactosidase, guanylate cyclase activator 2B, tripeptidylpeptidase 1, branched-chain amino acid aminotransferase, zinc transporter ZIP13, protein LEG1 homolog, and odour-binding protein 2 a.

Drawings

Figure 1 shows TH immunohistochemistry results; wherein A1 to A3 of FIG. 2 show controls 1-3; b1 to B4 show experimental groups 1-4; c of fig. 2 represents TH positive cell number statistics.

FIG. 2 shows the differential protein numbers at different time points in the α -Syn transgenic mice.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure.

Example 1 trans-clave behavioural assay of α -Syn (A53T) positive transgenic mice

4-week-old clean-grade male alpha-Syn (A53T) positive transgenic mice [ C57BL/6J-Tg (PDGF-alpha synuclein A53T) ILAS ] (n ═ 4) were purchased from the institute of Experimental animals, medical institute of Chinese academy of medicine, and the animal license was SCXK (Jing) 2014-0011. The animal experiments followed review and approval (animal welfare assurance number: ACUC-A02-2014-007). All animals were kept in a standard environment (room temperature 22 + -1 deg.C, humidity 65% -70%) with 12 hours light and dark cycles.

Rotarod behavioural test was used to evaluate the mouse limb motor coordination ability. The rotation speed of the rod rotating instrument (developed by the institute of medicine of Chinese academy of medicine) is respectively set to be constant speed 25rpm, 30rpm and 35rpm, and each time interval is 30min and time limit is 2 min. The time when the mouse first fell off the rod was recorded as latency, indicating its motor coordination ability.

Before the experiment is started, the mouse is placed in a kinematic laboratory environment for more than 30min, short-term influence on the mouse caused by changes of carrying, lighting and the like is avoided, and the mouse is trained for 1min at the rotating speed of 15rpm to adapt to a rod rotating instrument.

Data are presented as mean ± SEM, and differences between groups were analyzed using the T-test, with P <0.05 as statistically different.

The results of rotarod ethology tests show that the 2-month-old alpha-Syn transgenic mice are taken as a control, the mouse ethology has obvious difference at 6 months, namely, obvious dyskinesia is shown, and the dyskinesia is approximately consistent with that of previous researches (see Gaoning, establishment of PDGF-h-alpha-Synuclein transgenic Parkinson disease mouse model, 2008, university of Chinese cooperative medical science; Wen, research on the effect of heat shock protein 70 on inhibiting astrocyte-mediated inflammation in the research on improving the dyskinesia of alpha-Synuclein transgenic mice by the compounds WYD1-8 and WYD7-6, 2016, Beijing cooperative medical college) about 5 months. The reason why the difference in the behavioural characteristics was not observed at 9 months or 10.5 months was presumably that the number of experimental animals was small. (Table 1)

Table 1: drop latency of alpha-Syn transgenic mice at different time points

Example 2 immunohistochemical staining of α -Syn (A53T) positive transgenic mice

Immunohistochemical staining was performed after the last behavioral test. Age-matched male C57BL/6 mice were used as controls (n-3).

After anesthetizing the mice, the mice were perfused with normal saline and then 4% paraformaldehyde, and the isolated brains were fixed in 4% paraformaldehyde for 24h, then transferred to a 4% paraformaldehyde solution containing 30% sucrose and dehydrated until the brain tissue sinks to the bottom. The brain tissue was removed, dehydrated, embedded and sectioned to a thickness of 4 μm. Dewaxing to water, placing the slices in EDTA buffer solution for microwave repair, washing with PBS buffer solution, adding 3% hydrogen peroxide solution, incubating at room temperature for 10min, washing with PBS buffer solution, adding 5% BSA for sealing for 20min, incubating with TH (1: 100) antibody at 4 deg.C overnight, and adding secondary antibody for incubating for 50 min. Adding a freshly prepared DAB solution for color development, and adding purified water to stop the color development reaction. Hematoxylin counterstaining, 1% hydrochloric acid alcohol differentiation for 1s, tap water washing, ammonia water returning to blue, and running water washing. Slicing with gradient ethanol (70-100%) for 1min, dehydrating, drying, removing xylene, and sealing with neutral gum. And (4) framing scanning. The number of TH (tyrosine hydroxylase) immunopositive cells in the substantia nigra region of the brain in mice was recorded. All evaluations were done by researchers unfamiliar with the experimental design.

TH is the rate-limiting enzyme for dopamine synthesis, and alpha synuclein inclusion bodies are usually co-localized with TH, so the number of TH positive neurons can reflect the condition of dopaminergic neuron loss. Immunohistochemical results showed a general trend of decreased numbers of TH positive cells in the experimental group, i.e. loss of dopaminergic neurons. No significant differences were shown, which may be related to individual differences and fewer samples. (FIG. 1)

Example 3 urine collection and sample preparation

Urine samples of A53T mice at months 2, 4, 6, 8 and 10 were collected, respectively, and the urine sample collected at month 2 was used as a control group. Mice were placed individually in metabolic cages overnight (12h) to collect urine samples. During the urine collection period, food is not provided, and water can be freely drunk, so that the urine pollution is avoided.

The collected urine was centrifuged at 3000 Xg for 10min at 4 ℃ to remove cell and particulate matter precipitate, and the supernatant was stored at-80 ℃. Prior to urine protein extraction, urine samples were centrifuged at 12000 Xg for 10min at 4 ℃ to remove cell debris. The supernatant was precipitated at 4 ℃ for 12h using four times the volume of pre-cooled ethanol. The above samples were centrifuged at 12000 Xg for 10min at 4 ℃. The pellet was resuspended in lysis buffer (8mol/L urea, 2mol/L thiourea, 25mmol/L DTT and 50mmol/L Tris). Protein concentration was determined using the Bradford method.

Using a Filter-aided protein hydrolysis method (FASP) (see also

J.R. et al, Universal sample preparation method for protein analysis. Nature Methods,2009.6(5): p.359-362) applied membrane-assisted enzymatic digestion to urine proteins. 100ug of protein per sample was applied to a 10kDa filter (Pall, Port Washington, N.Y., USA). The protein sample was washed by sequentially adding UA (8mol/L urea, 0.1mol/L Tris-HCl, pH 8.5) and 50mmol/L NH4HCO3 solution. Adding 20mmol/L DTT reduced protein (37 deg.C, 1h), reacting 50mmol/L IAA away from light for 30min to alkylate the protein disulfide bond. According to the mass of the protein: trypsin ((Trypsin Gold, Promega, Fitchburg, WI, USA) was added at 1: 50 enzyme mass, incubated overnight at 37 ℃ the peptide fragments were collected by centrifugation, desalted using Oasis HLB solid phase extraction column (Waters, Milford, MA), dried by vacuum concentrator (Thermo Fisher Scientific, Bremen, Germany) and stored at-80 ℃.

The mixed peptide fragment samples were fractionated using high pH reverse phase polypeptide fractionation. The mixed peptide fragment samples were loaded onto rotating small columns using a high pH reverse phase polypeptide fractionation kit (84868, Thermo Fisher, USA). The bound peptide was eluted with a gradient of increasing acetonitrile concentration for a total of 10 fractions, including 1 mobile fraction, 1 eluted fraction and 8 gradient sample fractions. The fractionated sample was dried using a vacuum concentrator and redissolved in 20. mu.L of 0.1% formic acid water.

Example 4LC-MS/MS analysis

1) DDA database collection of component fractionated samples

And (3) carrying an Orbitrap Fusion Lumos high resolution mass spectrometer to collect data of the graded peptide fragment sample by using EASY-nLC 1200 ultra performance liquid chromatography. The peptide fragment dissolved in 0.1% formic acid water was loaded onto a pre-column (75 μm × 2cm,3 μm, C18,100A °), and the eluate was loaded onto a reverse phase analytical column (50 μm × 250mm,2 μm, C18,100A °) with an elution gradient of 5-30% mobile phase B (99.9% acetonitrile, 0.1% formic acid, flow rate of 0.3 μ L/min), 60 min. To achieve fully automatic, sensitive signal processing, a calibration kit (iRT kit, Biognosys, Switzerland) was used in all samples at a concentration of 1:20 v/v.

10 fractions were analyzed in DDA-MS mode with the following parameter settings: orbitrap full scan at 60000, 350 and 1550m/z primary resolution, cycle time 3s (highest speed mode), Automatic Gain Control (AGC) 1e6, and maximum injection time 50 ms. The secondary scan was performed in Orbitrap with a resolution of 15000, a screening window of 2Da and a collision energy of 32% (HCD). The AGC target is 5e4, and the maximum sample injection time is 30 ms.

2) DIA data acquisition of experimental samples

20 experimental samples were analyzed in DIA-MS mode. Liquid phase parameters were collected from the DDA database. The mass spectrum parameters were set as follows: carrying out primary full scanning at 60000 resolution and 350-; a second scan was then performed at a resolution of 30000, 36 screening windows were established, HCD collision energy was 32%, AGC target 1e6, and maximum injection time was 50 ms. The window calculation mode is as follows: and (3) sequencing all the identified peptide fragment quantities according to m/z by using DDA library searching results collected by library building, and dividing into 36 groups, wherein the m/z range of each group is the window width for collecting DIA data.

3) Data analysis

The component samples were data processed using a protome scanner (version 2.3, Thermo Scientific, Germany) to construct a database. The library was searched using the SwissProt mouse database (5 month update 2019, containing 17016 protein sequence information) with an additional iRT peptide sequence. The search parameter settings are as follows: the mass deviation of the parent ions is 10ppm, and the mass deviation of the fragment ions is 0.02 Da; the fixed modification was ureidomethylation for cysteine (+58.00Da) and the variable modification was oxidation for methionine (+15.995Da), with the others set as default parameters. The False Discovery Rate (FDR) at the protein and peptide levels was set to 0.01. The library results were imported into a Spectronaut Pulsar (Biognosys, Switzerland) to create a library of spectra: 665 proteins, 29028 secondary spectra.

The DIA-MS raw file is imported into Spectronaut Pulsar using default settings. Quantification was based on all secondary fragment ion peak areas. And identifying the high-reliability protein by identifying at least 2 specific peptide fragments of each protein according to the peptide fragment level q value of less than 0.1. The screening criteria for differential proteins were: p value <0.01, fold change in protein abundance > 2.

4) Changes in urine proteome

Differentially expressed protein screening criteria: the protein abundance change times are more than 2 times, and the p value is less than 0.01. Simultaneously, the Uniprot database is used for searching human homologous proteins corresponding to the proteins.

A total of 513 highly reliable proteins were identified by quantitative DIA analysis. Through screening, 17 kinds of human homologous differential proteins are identified in comparison with the 2 nd month, wherein the 4 th month comprises 2 kinds of differential proteins, 1 kind of up-regulation and 1 kind of down-regulation; at 6 months, 7 different proteins are added, 4 are up-regulated and 3 are down-regulated; at month 8, 5 different proteins were present, 1 up-regulated and 4 down-regulated; there were 10 different proteins, 5 up-regulated and 5 down-regulated at month 10. There was no continuous change in protein. (see FIG. 2, Table 2).

Table 2: differential proteins of alpha-Syn transgenic mice

5) Differential protein analysis

The unique differential proteins exist in the alpha-Syn transgenic mice at different time points, and the urine proteome has great potential to reflect the disease process of the A53T mice, and the specific analysis is as follows.

At month 4, 2 different proteins were significantly changed before no significant difference occurred in the behavior and pathology, and 1 of them was reported to be associated with parkinson's disease. Actin (Actin) is involved in various types of cell motility, and the interaction of alpha synuclein with Spectrin triggers pathological changes in the Actin cytoskeleton, as verified in studies on alpha-Syn transgenic mice, see Ordonez, D.G., M.K.Lee, and M.B.Feany, alpha-synuclein inductors Mitochondrial dye function through spectra and the active cytoleton.Neuron, 2018.97(1): p.108-124.e 6.

At 6 months, dyskinesia just appeared, and there were 7 distinct changes in protein, of which 4 were reported to be directly or indirectly related to parkinson's disease, and 3 were continuously changed at 6, 8 and 10 months, namely, mature protein-2 (Formin-2), Splicing factor 3A subunit 1(Splicing factor 3A subunit 1), and Isopentenyl diphosphate isomerase Delta-isomerase 1(Isopentenyl-diphosphate Delta-isomerase 1). Protein-2 (Formin-2) is highly expressed in the developing adult central nervous system, see Leader, B.and P.Leder, Formin-2, a novel for homology protein of the cauluccino subfamily, is high expressed in the devising and adut central nervous system. Mech Dev,2000.93(1-2): p.221-31, as actin-binding protein, involved in the assembly and recombination of the actin cytoskeleton, see Pfunnder, S.et al, spiral-type actin nuclei polynucleotides with Formin-2to drive asymmetry synthesis cell division. Current Biol,2011.21(11): p.955-60; azoury, J.et al, Spindle position in mice houses on a dynamic market of action files. curr Biol,2008.18(19): p.1514-9. In addition, it has been found that it is down-regulated in the brain tissue of patients with Alzheimer's disease, see, for example, Agis-Balboa, R.C. et al, Formin 2links neuropsychiatric phenols at young age to an involved rare ridge for the evaluation of Embo j,2017.36(19): p.2815-2828; isopentenyl diphosphate isomerase Delta-isomerase 1(Isopentenyl-diphosphate Delta-isomerase 1) is one of two isoforms of Isopentenyl Diphosphate Isomerase (IDI) in humans (IDI) (IDI1 and IDI2), IDI1 and IDI2 may be involved in the production of cholesterol metabolites in neurons, leading to aggregation of alpha synuclein during Lewy body formation, see Nakamura, k. et al, Isopentenyl diphosphite isomerase, alpha cholestrosterol synthesis enzyme, localized in Lewy bodies neuro, 2015.35(5): p.432-40; thioredoxin-dependent peroxide reductase (mitochondrial) is involved in antioxidant mechanisms and upregulated in the striatal tissues of 6-OHDA induced Parkinson animal models, see Lessner, G.et al, Differential proteins of the striatal from chemical industries display visual structural modification processes.J. Proteome Res,2010.9(9): p.4671-87; elongation factor 1-alpha 1(Elongation factor 1-alpha 1) is co-immunoprecipitated with human LRRK2, while autosomal dominant mutations in leucine-rich repeat kinase 2(LRRK2) are the most common genetic cause of Parkinson's disease, see Gilardon, F., Interaction of Elongation factor 1-alpha with expression-rich repeat kinase 2 enzymes activity and microtube bundling in vision. neuroscience,2009.163(2): p.533-9.

At month 8, there were 5 significant changes in the differential protein, of which 3 were reported to be directly or indirectly associated with parkinson's disease. In addition to the mature protein-2 (Formin-2), Isopentenyl diphosphate isomerase Delta-isomerase 1(Isopentenyl-diphosphate Delta-isomerase 1), the concentration of the isoform of Complement factor B (complementary factor B) is lower in the cerebrospinal fluid of Parkinson's disease patients than in normal subjects, and the isoform of Complement protein in the cerebrospinal fluid may be a biomarker of neurodegenerative diseases, see Finehout, E.J., Z.Franck, and K.H.Lee, complementary protein isoforms in CSF among porous biomakers for neuro-egenerative disease Markers,2005.21(2): p.93-101.

At month 10, there were 10 significant changes in the differential protein, of which 6 were reported to be directly or indirectly associated with parkinson's disease. In addition to Formin-2 (Formin-2), Isopentenyl-diphosphate isomerase Delta-isomerase 1(Isopentenyl-diphosphate Delta-isomerase 1), complement factor B (complementary factor B), Zinc transporter ZIP13(Zinc transporter ZIP13) Zinc transporter dysfunction is associated with the dynamic balance of Zinc ions present in Parkinson's disease, see Rolfs, A.and M.A.Hediger, Metal ion transporters in mammals: structure, function and pathological mechanisms J. phosphor, 1999.518(Pt 1): p.1-12, whereas changes in Zinc transporter Expression and function may contribute to the pathogenesis of neurodegenerative diseases, see Leung, K.W. et al, Expression of ZnT transporters in neuronal in molecular weight, change in the function of Zn < 12 > -3. 1223. vitroplastic cells, Zn < 12 > 3. vitroplastic in brain cells, see changes in the function of the neuron, Zn < 12 > -3. 1223. and vitamin E < 3. 1223. cells, see Mocchegiani, E.et al, Brain, imaging and neuro-synthesis, roll of zinc availability, prog Neurobiol,2005.75(6): p.367-90; tripeptidyl peptidase 1(Tripeptidyl-peptidase 1) exhibits Parkinson's disease characteristics in 5 patients with TPP1mutation, see Di Giacopo, R. et al, characterized late amino peptide lipofectin to TPP1 events, Clinical, molecular and biochemical transformation in human diseases J Neurol Sci,2015.356(1-2): p.65-71; simonati, A. et al, A CLN2gene mutation is associated with polypeptide sulfate and dysstonia in LINCL. neuropedias, 2000.31(4): p.199-201; le, N.M.and S.Parikh, Late in facial nerve center lipofuscinosis and dopamine specificity. J Child Neurol,2012.27(2): p.234-7; the regulation of Branched-chain amino-acid aminotransferase (cytotoxic aminotransferase) in macrophage function has therapeutic significance in inflammatory diseases, see Patathanassiu, A.E. et al, BCAT1control metabolic remodeling in activated human mammograms and is associated with inflammatory with diseases Nat Commun,2017.8: p.16040, whereas chronic, acute central nervous system inflammation is responsible for the damage to the nervous system of Parkinson's disease, see Mazzio, E.A. et al, Nature product HTP screening for the activation of cytokine-induced nuclear molecular adhesion promoters (CINCCs) NO 2-LPS/IFOI activity, see BCAT 19. T19. T.19. for oxidative stress-mediated dysfunction of BCAT 19. T19. T.19. T.8. T. related to oxidative stress-mediated by BCAT 19. T19. T. J. for oxidative stress promotion of Parkinson's disease, m. et al, BCAT1 proteins cell promotion through amino acid catalysis with type IDH1. nat. Med,2013.19(7): p.901-908.

Comparing the 17 differential proteins with aging biomarkers (Li, X.and Y.Gao, Potential real imaging markers of 20-month-old rates. PeerJ,2016.4: p.e2058) under the conditions that the abundance change multiple is more than 2 times and the p value is less than 0.01, the same differential protein is not found, which indicates that the change of disease progression of the alpha-Syn transgenic mouse is far greater than that of aging, namely, the 17 differential proteins screened at this time are more related to the simulated Parkinson disease of the alpha-Syn transgenic mouse.

6) Analysis of biological function of differential proteins

Randomly grouping samples of each time point according to different change multiples and p values respectively to find a group of screening conditions with the lowest false positive rate, and considering that differential proteins obtained by screening under the conditions are more reliable. Generally, the more stringent the selection conditions, the lower the false positive rate, but the more stringent the selection conditions will also result in less differential protein being obtained and false negative protein being screened out. Because of the few differential proteins, no assessment of the biological function annotation could be performed on the identified differential proteins using the online free software DAVID 6.8(https:// DAVID. ncifcrf. gov /), and therefore, the experiment was performed on the differential proteins screened under the conditions that the fold change was greater than 1.5 and the p-value was less than 0.05 for the biological function complementation analysis. (Table 3)

The differential proteins were functionally annotated using DAVID, displaying information about the biological processes, cellular composition, and molecular function of the differential proteins at each time point. Pathway analysis was performed on the differential protein using IPA.

It was found that inflammatory responses and Vitamin D metabolism began at month 8, whereas chronic and acute CNS inflammation was responsible for the damage to the nervous system of Parkinson's disease, as assessed by biofunctional annotations, see Mazzio, E.A. et al, Natural product HTP screening for the assessment of cytokine-induced neurophil chemical attack (CINCs) and NO2-in LPS/IFNgamma activated dietary cells, J Neuroimemunol, 2017.302: p.10-19, and additionally, Vitamin D is involved in the regulation of calcium, phosphate metabolism, immune responses and Brain development, reported to be a good candidate serum biomarker for Parkinson's disease and Alzheimer's disease, see Bivona, G.et al, Non-Skelet al, Vitamin D: phosphor metabolism, protein metabolism to protein metabolism (Pathology 2019.55). Analysis of cellular composition revealed that the differential proteins were secreted proteins, mostly from extracellular matrix and membranes. For molecular functions, it was shown that most of the proteins had enzymatic activity.

Pathway analysis shows that pathways at different time points have respective characteristics, and are partially reported to be related to Parkinson's disease. For example, inflammatory responses such as Leukocyte Extravasation Signaling (Leukocyte Extravasation Signaling) and Tight Junction Signaling (light Junction Signaling), endocytosis are reported to be the two most likely pathways associated with Sporadic Parkinson's Disease at month 4, Hu, Y, et al, and also, adolling Genome-Wide Association Study a Pathway for systemic delivery of fungal Parkinson's Disease, 2016.53(7): 4302-18, Remodeling of Epithelial adhesive Junctions (remodelling of Epithelial adhesives Junctions), lectin interaction at Neuromuscular Junctions (Agrin Interactions at neuro-common Junction) is the second most affected pathway in Parkinson's disease, see Karim, S. et al, Gene expression analysis from Epithelial to Epithelial porous links, park person's disease, cancer and cardiac disease, CNS neural disease d Drug Targets,2014.13(8): p.1334-45. Acute Phase Response Signaling (Acute Phase Response Signaling) and various signals related to oxidative stress appear from month 6, which suggests that there may be a certain relationship between Parkinson's disease and immune diseases

Table 3: differential protein of alpha-Syn transgenic mice (p < 0.05)

The results of this study show that urine proteome can reflect changes in disease progression of α -Syn transgenic mice. Before pathological and behavioral changes do not appear, the differential protein with obvious changes can be identified in urine. Continuously changed differential proteins and respective unique differential proteins exist at different time points, more than half of the differential proteins are reported to be directly or indirectly related to the Parkinson's disease, the urine has great potential for early diagnosis and development of the Parkinson's disease, and the urine proteome research of the Parkinson's disease has great potential. Meanwhile, different combinations of the markers can provide new ideas and clues for early diagnosis of neurodegenerative diseases, and the method has a value for developing large-scale clinical research.

Claims (8)

1. Use of an agent for detecting the amount of protein in urine of a subject in the manufacture of a medicament for diagnosing parkinson's disease in said subject, wherein the protein in said urine is a combination of: protein TOPAZ1, actin, elongation factor 1- α 1, thioredoxin-dependent peroxiredoxin reductase, isopentenyl diphosphate isomerase δ -isomerase 1, splicing factor 3A subunit 1, PDZK1 interacting protein 1, proteogen-2, complement factor B, α -N-acetylgalactosase, guanylate cyclase activator 2B, tripeptidylpeptidase 1, branched-chain amino acid aminotransferase, zinc transporter ZIP13, protein LEG1 homolog and odour binding protein 2 a.

2. The use of claim 1, wherein the diagnosis is an early diagnosis defined as the subject having no apparent behavioral abnormalities.

3. The use of claim 1 or 2, wherein the reagent for detecting the amount of protein in the urine of a subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof.

4. The use of claim 3, wherein the reagent for detecting the protein content in urine of a subject is a monoclonal antibody.

5. The use of claim 1 or 2, wherein the subject is a mammal.

6. The use of claim 5, wherein the subject is a human.

7. The use of claim 1 or 2, wherein an increase in actin, elongation factor 1-alpha 1, thioredoxin-dependent peroxide reductase, PDZK1 interacting protein 1, alpha-N-acetyl galactosidase, guanylate cyclase activator 2B, tripeptidyl peptidase 1, branched amino acid aminotransferase, zinc transporter ZIP13, odor binding protein 2a content, and/or a decrease in the content of protein TOPAZ1, isopentenyl diphosphate isomerase delta-isomerase 1, splicing factor 3A subunit 1, proteogen-2, complement factor B, protein LEG1 homolog, as compared to a healthy control, indicates that the subject has parkinson's disease.

8. A kit or chip for early diagnosis of parkinson's disease, comprising reagents for detecting the content of a set of proteins in the urine of a subject, wherein the set of proteins in the urine is a combination of: protein TOPAZ1, actin, elongation factor 1- α 1, thioredoxin-dependent peroxiredoxin reductase, isopentenyl diphosphate isomerase δ -isomerase 1, splicing factor 3A subunit 1, PDZK1 interacting protein 1, proteogen-2, complement factor B, α -N-acetylgalactosase, guanylate cyclase activator 2B, tripeptidylpeptidase 1, branched-chain amino acid aminotransferase, zinc transporter ZIP13, protein LEG1 homolog and odour binding protein 2 a.

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