Application of N-glycosylated protein assemblages in urine as targets in the diagnosis of preclinical and MCI stages of Alzheimer's disease

Abstract

This invention discloses the application of a combination of N-glycosylated proteins in urine as targets in the diagnosis of preclinical and MCI-stage Alzheimer's disease. The combination of N-glycosylated proteins in urine described in this invention is applicable to the early diagnosis of Alzheimer's disease, including the preclinical and MCI stages, and has the advantages of being non-invasive and accurate, with extremely high diagnostic specificity and sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnostic biomarker technology, specifically relating to the application of a combination of N-glycosylated proteins in urine as a target in the diagnosis of preclinical and MCI-stage Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that primarily affects the elderly. It is an age-related central nervous system degenerative disease characterized by progressive memory and cognitive impairment. Major clinical symptoms include memory loss, aphasia, agnosia, and executive dysfunction. The lesions in AD are concentrated in the hippocampus and cerebral cortex, resulting in diffuse cortical atrophy, weight loss, narrowing of the gyri, and widening of the sulci in the cerebral hemispheres. Often, lesions and clinical symptoms are already present in the brain of early-stage AD patients, and the affected areas worsen with age, leading to impaired brain function and an inability to improve bodily functions. In 1906, German neuropathologist Alois Alzheimer first reported this disease, describing the pathological features found in the brains of patients. These included neurofibrillary tangles (NFTs) composed of highly phosphorylated microtubule-associated protein (Tau) within nerve cells, and amyloid plaques formed by the aggregation and deposition of β-amyloid protein (Aβ) outside the cells. Subsequently, neurofibrillary tangles and amyloid plaques became two important pathological features for diagnosing Alzheimer's disease.

[0003] Alzheimer's disease primarily affects people over 65 years of age, with an estimated incidence rate of about 10% in this group and approximately 50% in those over 85. Alzheimer's patients account for 60%–80% of all dementia cases. Clinical studies indicate that AD can be divided into common, sporadic AD (SAD) and a minority (approximately 15%–20%) familial AD (FAD) with a family history. Common sporadic AD mainly affects people over 65, and its incidence increases with age. Therefore, research into the pathogenesis of AD and drug development are increasingly important for human health.

[0004] Current treatments for Alzheimer's disease (AD) are effective, but the limited number of drugs can only slow the progression of the disease. There are currently no drugs that can reverse the pathological progression of AD. Therefore, it is particularly important to develop accurate, non-invasive, and convenient diagnostic biomarkers and diagnostic kits for the early diagnosis of AD. Summary of the Invention

[0005] As is known to those skilled in the art, glycosylation is the binding of glycans to specific amino acid residues in proteins under the action of glycosyltransferases. It is an important post-translational modification of proteins, playing a crucial role in the growth, development, and survival of organisms. The main types of glycosylation are N-linked and O-linked glycosylation. N-linked glycosylation accounts for 90% of all glycosylations and is the most prevalent type. N-glycans are covalently linked to the asparagine radical (-NH2) of the protein, and the specific amino acid sequence is AsnXSer / Thr, where X is any amino acid except proline. N-glycosylated glycan synthesis begins in the endoplasmic reticulum and is completed in the Golgi apparatus. Its glycan composition is rich in mannose, and the terminal branches are rich in fucose. O-glycans are covalently linked to the serine or threonine radical (-OH) of the protein. O-glycosylation sites do not have conserved sequences, and the glycans do not have a fixed core structure; they can be a monosaccharide or a large polysaccharide. Dysregulation of glycosylated proteins affects multiple biological pathways in the progression of Alzheimer's disease (AD), including extracellular matrix dysfunction, neuroinflammation, synaptic dysfunction, altered cell adhesion, lysosomal dysfunction, endocytic transport regulation disorders, endoplasmic reticulum dysfunction, and cell signaling regulation disorders. Dysregulation of glycosylation, especially N-glycosylated proteins, has a significant impact on the development and progression of the disease, but related research in AD is still relatively lacking.

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of N-glycosylated protein combinations in urine as targets in the diagnosis of preclinical and MCI stages of Alzheimer's disease.

[0007] The technical solution of the present invention is as follows:

[0008] The application of a combination of N-glycosylated proteins in urine as targets in the preparation of diagnostic kits for early Alzheimer's disease. The combination of N-glycosylated proteins in urine includes N-glycosylated GALNS in urine and optional N-glycosylated proteins. The optional N-glycosylated proteins are at least one of N-glycosylated ICAM3, N-glycosylated TYRO3, and N-glycosylated CILP2. The aforementioned early Alzheimer's disease refers to preclinical Alzheimer's disease and MCI stage Alzheimer's disease.

[0009] Those skilled in the art will know that:

[0010] The preclinical stage, which may last for years or decades, begins with pathological changes in the hippocampus and medial temporal cortex. Abnormal Aβ aggregation forms jagged amyloid plaques, while tau protein phosphorylation and polymerization form neurofibrillary tangles. These pathological changes gradually spread to other related areas of the cortex until the excessive production and accumulation of Aβ in the brain reaches a critical level, triggering an amyloid cascade reaction. The criteria for the preclinical population are: 1. PET-verified abnormal Aβ aggregation forming jagged amyloid plaques; 2. PET-verified tau protein phosphorylation and polymerization forming neurofibrillary tangles; 3. Subjective cognitive decline, whether present or absent, but not meeting the cognitive decline criteria measured by various cognitive-related scales.

[0011] MCI (Mild Cognitive Impairment) stage is characterized by early pathology, ranging from mild neurotrophic disorder to early Braak pathology, and can persist for several years depending on individual resilience and brain reserve. Amyloid plaques and neurofibrillary tangles continue to accumulate and extend to more extensive areas of the cortex. Neuronal loss and synaptic dysfunction begin to appear, leading to mild memory decline. The criteria for selecting individuals for MCI stage are: a decline in cognitive levels as measured by various cognitive-related scales.

[0012] In a preferred embodiment of the present invention, when the early Alzheimer's disease is preclinical Alzheimer's disease, the optional N-glycosylated protein is N-glycosylated ICAM3 and N-glycosylated TYRO3.

[0013] In a preferred embodiment of the present invention, when the early Alzheimer's disease is MCI stage Alzheimer's disease, the optional N-glycosylated protein is N-glycosylated CILP2.

[0014] An early Alzheimer's disease diagnostic kit includes reagents capable of detecting a combination of N-glycosylated proteins in urine, the combination of which includes N-glycosylated GALNS in urine and optional N-glycosylated proteins, the optional N-glycosylated proteins being at least one of N-glycosylated ICAM3, N-glycosylated TYRO3, and N-glycosylated CILP2, wherein the early Alzheimer's disease is preclinical Alzheimer's disease and MCI stage Alzheimer's disease.

[0015] In a preferred embodiment of the present invention, when the early Alzheimer's disease is preclinical Alzheimer's disease, the optional N-glycosylated protein is N-glycosylated ICAM3 and N-glycosylated TYRO3.

[0016] In a preferred embodiment of the present invention, when the early Alzheimer's disease is MCI stage Alzheimer's disease, the optional N-glycosylated protein is N-glycosylated CILP2.

[0017] In a preferred embodiment of the invention, an enrichment component for enriching N-glycosylated proteins in urine is also included.

[0018] More preferably, the enrichment component is a ZIC-HILIC micropillar.

[0019] In a preferred embodiment of the present invention, the reagent includes LC-MS / MS analytical reagents.

[0020] The beneficial effects of this invention are: the combination of N-glycosylated proteins in urine in this invention is applied to the early diagnosis of Alzheimer's disease, including the preclinical and MCI stages, and has the advantages of being non-invasive and accurate, and has extremely high diagnostic specificity and sensitivity. Attached Figure Description

[0021] Figure 1 This diagram illustrates the basic situation of urinary N-glycosylation protein profile analysis in the control group, the preclinical group of Alzheimer's disease, and the MCI group in Example 1 of this invention.

[0022] Figure 2 This is a technical roadmap for screening urine N-glycosylated protein profiles of control populations, preclinical Alzheimer's disease patients, and patients in the MCI stage of Alzheimer's disease in Example 1 of the present invention to identify combinations of urine-specific N-glycosylated proteins from these populations.

[0023] Figure 3 This figure shows the experimental results of Western blot analysis of the glycosylation modification of specific N-glycosylated protein combinations in the urine of Alzheimer's disease preclinical and MCI stage patients and control groups in Example 1 of this invention.

[0024] Figure 4 This figure shows the experimental results of Example 1 of the present invention, which verified the specificity and sensitivity of the combination of N-glycosylated proteins specific to the preclinical and MCI stages of Alzheimer's disease for the diagnosis of the preclinical and MCI stages of Alzheimer's disease. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0026] Example 1

[0027] 1.1 Sample Source

[0028] The samples in this embodiment were 10 mL urine samples from control groups, preclinical patients, and patients in the MCI stage who had passed medical ethics review.

[0029] (1) Inclusion and exclusion criteria for the control group:

[0030] A. The inclusion criteria are as follows:

[0031] ① Age ≥ 55 years;

[0032] ②Elementary school education or above;

[0033] ③ No gender restrictions;

[0034] ④ADL (Activities of Daily Living) score ≤ 20 points;

[0035] ⑤CDR (Clinical Cognitive Impairment Scale) = 0 points;

[0036] ⑥ According to the standards of standardized research, the Mini-Mental State Examination (MMSE) was used for scoring. Those with a score above the threshold (more than 26 points for those with primary school education or above, and more than 25 points for those over 80 years of age) were included.

[0037] B. Exclusion criteria include:

[0038] ① History of severe head trauma;

[0039] ② History of drug or alcohol abuse;

[0040] ③ History of mental illness such as schizophrenia or other affective disorders (including affective disorders, schizophrenia, alcohol or drug abuse or dependence, etc.);

[0041] ④ Serological tests were used to rule out low levels of Vitamin B12, low levels of folic acid, and hypothyroidism.

[0042] ⑤ No history of cognitive impairment;

[0043] ⑥ No cerebral infarction, cerebral softening, or other space-occupying lesions were found on the plain MRI scan of the head.

[0044] (2) Inclusion and exclusion criteria for preclinical Alzheimer's disease patients:

[0045] A. According to the diagnostic criteria proposed by the National Institute on Aging and the Alzheimer's Disease Association, the preclinical stage of AD is generally divided into three phases: asymptomatic cerebral amyloidosis, amyloid protein positivity + synaptic dysfunction and / or early neurodegeneration, and amyloid protein positivity + evidence of neurodegeneration + very mild cognitive decline (as shown in Table 1 below). This diagnostic criterion emphasizes the value of biomarkers in the preclinical stage of AD.

[0046] Table 1

[0047]

[0048] B. According to the diagnostic criteria of the 2007 IWG and the 2014 IWG-2, the preclinical stage of Alzheimer's disease (AD) is usually divided into the asymptomatic risk period and the pre-symptomatic period. The asymptomatic risk period refers to patients who have objective evidence of cerebral amyloidosis and neurodegeneration through imaging or biomarkers, but whose clinical manifestations and neuropsychological examinations have not yet met the criteria for mild cognitive impairment (MCI). Although the pathophysiological process of AD has begun in the brain of these patients, they will not necessarily progress to AD in the end. The pre-symptomatic period refers to patients who carry autosomal dominant pathogenic gene mutations such as PSEN1, PSEN2, APP, or other pathogenic genes, but have not met the diagnostic criteria for MCI. These patients will eventually develop AD.

[0049] C. Other inclusion and exclusion criteria:

[0050] a. Inclusion criteria include:

[0051] ① Age ≥ 50 years old;

[0052] ②Elementary school education or above;

[0053] ③ No gender restrictions;

[0054] ④ADL (Activities of Daily Living) score ≤ 20 points;

[0055] ⑤CDR (Clinical Cognitive Impairment Scale) = 0 points;

[0056] ⑥ According to the standards of standardized research, the Mini-Mental State Examination (MMSE) was used for scoring. Those with a score above the cutoff (>26 points for those with primary school education or above, and >25 points for those over 80 years of age) were considered to have a score above the cutoff.

[0057] b. Exclusion criteria include:

[0058] ① History of severe head trauma;

[0059] ② History of drug or alcohol abuse;

[0060] ③ History of mental illness such as schizophrenia or other affective disorders (including affective disorders, schizophrenia, alcohol or drug abuse or dependence, etc.);

[0061] ④ Serological tests were used to rule out low levels of Vitamin B12, low levels of folic acid, and hypothyroidism.

[0062] ⑤ No history of cognitive impairment;

[0063] ⑥ No cerebral infarction, cerebral softening, or other space-occupying lesions were found on the plain MRI scan of the head.

[0064] (2) Inclusion and exclusion criteria for MCI period:

[0065] A. Inclusion criteria include:

[0066] ① Age ≥ 50 years old;

[0067] ②Elementary school education or above;

[0068] ③ No gender restrictions;

[0069] ④ DSM-IV diagnostic criteria and exclusion criteria for MCI, among which,

[0070] The diagnostic criteria for DSM-IV are:

[0071] 1) Subjective feeling of memory decline;

[0072] 2) Objective examination reveals evidence of MCI, such as an MMSE score of 18-21 for illiteracy, 21-24 for primary school education, 25-27 for secondary school education, and a GDS score of 2-3;

[0073] 3) Decline in daily living skills and social functioning;

[0074] 4) HIS score ≤ 4, excluding cognitive decline caused by specific reasons;

[0075] 5) The course of illness lasts longer than 3 months;

[0076] 6) Does not meet the diagnostic criteria for dementia (CDR scale = 0.5 points).

[0077] Each case met the above six diagnostic criteria, and the diagnosis was made by experts from the geriatric medicine research group based on a comprehensive analysis of medical history, physical examination, and scale test results.

[0078] The exclusion criteria for DSM-IV are:

[0079] 1) Exclude individuals with a history of mental illness or congenital intellectual disability;

[0080] 2) Exclude patients with severe heart, lung, liver, or kidney dysfunction, severe endocrine system diseases, severe infectious diseases, and toxic encephalopathy;

[0081] 3) Exclude patients with neurological diseases that can cause brain dysfunction, such as stroke, Parkinson's disease, brain tumors, etc.

[0082] 4) Exclude patients with depression;

[0083] 5) Exclude individuals with a history of head trauma or taking certain medications;

[0084] 6) Exclude individuals who have been identified as alcohol or drug dependent within the past 6 months.

[0085] Table 2 below shows the basic information of the control population and early Alzheimer's disease patients (preclinical and MCI stages) obtained according to the above criteria for urinary N-glycosylation proteomic analysis.

[0086] Table 2

[0087]

[0088]

[0089] 1.2 Evaluation Criteria

[0090] Quality control was performed using 1% Total FDR (glycopeptide FDR at spectrum level).

[0091] 1.3 Experimental Apparatus

[0092] Orbitrap Fusion TM Lumos TM Tribrid TM Thermo Scientific TM OrbitrapFusion TM Lumos TM Tribrid TM Mass spectrometer.

[0093] 1.4 Glycosylated proteomics analysis

[0094] Glycosylation is the most prevalent and complex type of post-translational modification in living organisms. In eukaryotic cells, approximately 50% of proteins undergo glycosylation. Studying glycosylated proteomics is crucial for elucidating numerous life processes, such as immune responses, reproduction and development, pathogen infection, and tumorigenesis and progression. However, due to the small proportion of glycosylation in biological samples, the complex microscopic heterogeneity at the same glycosylation site, and the very poor ionization efficiency of glycosylated peptides (their signal is severely suppressed by unmodified peptides during ionization), specific enrichment of glycosylated proteins / peptides before mass spectrometry analysis is essential in glycosylated proteomics research. The two most common types of glycosylation are N-linked glycosylation and O-linked glycosylation. Among the many well-developed glycosylation enrichment techniques, boric acid enrichment and hydrophilic interaction chromatography (HILIC) are recognized as the two most universally applicable techniques that maintain the integrity of the glycan structure. The glycosylation proteomics research strategy in this embodiment is to use ZIC-HILIC microcolumns to specifically enrich glycosylated peptides in complex samples at the peptide level, followed by LC-MS / MS analysis.

[0095] 1.5N-glycosylated proteomics experimental methods (specific ideas are as follows) Figure 1 (As shown)

[0096] 1.5.1 Sample Preparation

[0097] Add lysis buffer (1% SDS, 8M urea, 1×Protease Inhibitor Cocktail (Roche Ltd.Basel, Switzerland)) to the sample to be lysed, shake and grind for 120s × 3, and lyse on ice for 30min. After high-speed centrifugation for 15min (15000rpm, 4℃), collect the supernatant.

[0098] 1.5.2 Proteolysis

[0099] The protein concentration in the supernatant was determined using a BCA protein assay kit. Subsequently, 1 mg of protein from each sample was transferred to a new EP tube and adjusted to 200 μL with 8 M urea. 20 μL of 0.5 M TCEP was added and the mixture was reacted at 37 °C for 1 hour, followed by the addition of 40 μL of 1 M iodoacetamide and incubation at room temperature in the dark for 40 min. Then, acetone pre-chilled at -20 °C was added at a sample:acetone ratio of 1:5, and the mixture was incubated overnight at -20 °C to precipitate. The sample was then centrifuged at high speed (12000 g, 20 min, 4 °C) and the supernatant was discarded. 1 mL of pre-chilled 90% acetone solution was added, and the sample was vortexed to wash it. The sample was then centrifuged again at high speed (12000 g, 20 min, 4 °C) and the supernatant was discarded. This washing step was repeated twice. After drying at room temperature until the acetone on the precipitate surface is completely evaporated, the precipitate is redissolved in 1 mL of 100 mMMTEAB. Trypsin (Promega, Madison, WI) is added at an enzyme:protein (mass ratio) of 1:50, and the mixture is incubated overnight at 37°C. After desalting using a C18 desalting column, the precipitate is analyzed using a peptide quantification kit (Pierce). TM 23275) The final concentration of the peptide was determined and lyophilized to obtain the peptide sample.

[0100] 1.5.3 Enrichment of Glycosylated Peptides

[0101] Approximately 30 mg of ZIC-HILIC chromatographic packing material (Merck Millipore, Merck KGaA, Darmstadt, Germany) was loaded onto a tip via a C8 disk to prepare a ZIC-HILIC microcolumn. The peptide sample obtained above, dissolved in 80% ACN and 1% TFA solution, was repeatedly loaded onto the ZIC-HILIC microcolumn to adsorb as many glycosylated peptides as possible. The microcolumn was then washed with 80% ACN and 1% TFA solution. The adsorbed glycosylated peptides on the ZIC-HILIC microcolumn were then eluted sequentially with 0.1% TFA solution, 25 mM NH4HCO3 solution, and 50% ACN solution. The eluates were combined and lyophilized to obtain the lyophilized peptide sample.

[0102] 1.5.4 Nano-HPLC-MS / MS Analysis

[0103] The lyophilized peptide samples were reconstituted in 10 μL of Solvent A (A: 0.1% FA in H2O) and then separated and identified using nanospray LC-MS / MS. The entire LC-MS system consisted of an EASY-nano-LC 1200 chromatography system (Thermo Fisher Scientific, MA, USA) tandem with an Orbitraq Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, MA, USA). 3 μL of sample was directly loaded, and separation was performed using a C18 analytical column (Acclaim PepMap, 15 cm x 75 μm id) with a linear gradient: 3% B to 32% B (B: 0.1% FA in ACN) over 180 min. The column flow rate was controlled at 300 nL / min, and the electrospray voltage was 2 kV. The Fusion mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition, with Stepped Collision Mode set to on. A full-scan spectrum (m / z 350-2000) was obtained at 120K quality resolution, with an AGC target of 500,000, a maximum injection time of 50ms, dynamic exclusion after n times (n=1), and a dynamic exclusion duration of 15s. Subsequent HCD MS / MS scans were performed at 15K resolution, with an isolation window of 4, detector type of Orbitrap, an AGC target of 500,000, a maximum injection time of 250ms, collision energy of 30%, stepped collision mode set to on, and an energy difference of ±10%.

[0104] 1.5.5 Data Analysis

[0105] Tandem mass spectrometry was performed using pGlyco 3.0 (pFind Studio), searching protein and glycan databases (pGlyco, species: human) with trypsin digestion. Search parameters included: fragment ion mass tolerance of 20 ppm, precursor ion mass tolerance of 10 ppm, maximum missed fragments of 2, fixed modification: Carbamidomethylation (C) 57.02, and variable modification: Oxidation (M) 15.99. Glycosylated peptides were filtered using 1% Total FDR quality control. Quantification was performed using pGlyQuant (pFind Studio) for label-free analysis.

[0106] 1.6 By analyzing the urinary N-glycosylation proteome results, specific combinations of urinary N-glycosylation proteins were screened from the control group, preclinical group, and MCI group as candidates for subsequent diagnostic biomarkers.

[0107] Analysis of urinary N-glycosylation proteome results identified urinary N-glycosylation-specific proteins in the control, preclinical, and MCI stages. These proteins were then combined as candidate diagnostic markers. The combination of GALNS and CILP2 was used in the preclinical and control groups, while the combination of GALNS, ICAM3, and TYPO3 was used in the MCI stage and control groups. The specific screening and combination process is as follows: Figure 2 Firstly, this embodiment uses ProteoSpin. TM The Urine Protein Concentration Midi Kit was used to extract urinary proteins from control individuals, individuals in the preclinical stage of Alzheimer's disease, and individuals in the MCI stage. Approximately 800 μg of protein was extracted, with a concentration of 0.8 μg / μL. In this example, Western blotting was performed on the urinary protein using lycopene as an internal control. Specific experimental results are as follows: Figure 3 As shown, in the normal control and preclinical groups: GALNS showed a single band in the normal population and a double band in the patient population; ICAM3 and TYPO3 showed a double band in the normal population and a single band in the patient population; in the normal control and MCI group: GALNS and CILP2 showed a single band in the normal population and a double band in the patient population, thus correlating the glycosylation modification of biomarker proteins with the gel electrophoresis bands.

[0108] 1.7 Validation of the specificity and sensitivity of the protein combinations screened in 1.6 using urine samples from control populations, preclinical patients, and patients in the MCI stage.

[0109] As shown in Table 3, a sex- and age-matched normal population served as the control group. The experimental group was selected based on a combination of clinical symptoms, patient complaints, cognitive scales, and Aβ / Tau PET-CT scans, identifying individuals in the preclinical stage (Toward defining the preclinical stages of Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease, 2011) and the mild cognitive impairment (MCI) stage (The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease, 2011). First, N-glycosylation protein profiles in the urine of the control, preclinical, and MCI populations were used to screen for proteins with specific N-glycosylation modifications in urine, serving as biomarkers for early diagnosis. Next, Western blot was used to clinically test a larger batch of urine samples from the three groups and the results were compared with clinical diagnoses to validate the selected biomarkers.

[0110] (1) Normal control and preclinical group:

[0111] GALNS: A single band in the normal population and two bands in the patient population indicate a true positive result; two bands in the normal population indicate a false positive result; a single band in the patient population indicates a false negative result.

[0112] ICAM3 and TYPO3: A double band in the normal population and a single band in the patient population indicate a true positive result; a single band in the normal population indicates a false positive result; and a double band in the patient population indicates a false negative result.

[0113] The ROC curve was calculated to assess the accuracy of biomarkers for preclinical diagnosis.

[0114] (2) Normal control and MCI stage group:

[0115] GALNS and CILP2: A single band in the normal population and two bands in the patient population indicate a true positive result; two bands in the normal population indicate a false positive result; a single band in the patient population indicates a false negative result.

[0116] The ROC curve was calculated to assess the accuracy of biomarkers for preclinical diagnosis.

[0117] By performing ROC analysis on the comparative results and analyzing the area under the curve, this embodiment found that the specific urinary N-glycosylated protein combinations in normal controls, preclinical stages, and MCI stages have extremely high diagnostic specificity and sensitivity. Specific results are as follows: Figure 4 As shown.

[0118] Table 3 below shows the basic information of the control population and Alzheimer's disease patients used in this section to verify the specificity and sensitivity of the Alzheimer's disease-specific N-glycosylated protein combination for diagnosing Alzheimer's disease. (This is completely different from the control population and early-stage Alzheimer's disease patients in Table 2.)

[0119] Table 3

[0120]

[0121] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of a combination of N-glycosylated proteins in urine as targets in the preparation of early Alzheimer's disease diagnostic kits, characterized by: When early Alzheimer's disease is preclinical Alzheimer's disease, the combination of N-glycosylated proteins in the urine includes N-glycosylated GALNS, N-glycosylated ICAM3, and N-glycosylated TYRO3; when early Alzheimer's disease is MCI stage Alzheimer's disease, the combination of N-glycosylated proteins in the urine includes N-glycosylated GALNS and N-glycosylated CILP2.

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