Application of N-glycosylated protein assemblages in urine as targets in the early diagnosis of Alzheimer's disease

Abstract

This invention discloses the application of a combination of N-glycosylated proteins in urine as targets in the early diagnosis of Alzheimer's disease. The combination of N-glycosylated proteins in urine described in this invention, when applied to the early diagnosis of Alzheimer's disease, has the advantages of being non-invasive and accurate, and exhibits extremely high specificity and sensitivity for early diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnostic biomarker technology, specifically relating to the application of N-glycosylated protein combinations in urine as targets in the early diagnosis of 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 the application of N-glycosylated protein combinations in urine as targets in the early diagnosis 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 an early diagnostic kit for Alzheimer's disease is characterized in that the combination of N-glycosylated proteins in urine includes N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1 in urine.

[0009] In a preferred embodiment of the present invention, the N-glycosylated protein group in urine consists of N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1 in urine.

[0010] An early diagnostic kit for Alzheimer's disease includes reagents capable of detecting a combination of N-glycosylated proteins in urine, including N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1.

[0011] In a preferred embodiment of the present invention, the N-glycosylated protein group in urine consists of N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1 in urine.

[0012] In a preferred embodiment of the invention, an enrichment component for enriching N-glycosylated proteins in urine is also included.

[0013] More preferably, the enrichment component is a ZIC-HILIC micropillar.

[0014] In a preferred embodiment of the present invention, the reagent includes LC-MS / MS analytical reagents.

[0015] 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, which has the advantages of being non-invasive and accurate, and has extremely high specificity and sensitivity for early diagnosis. Attached Figure Description

[0016] Figure 1 This is a basic diagram showing the analysis of urinary N-glycosylation protein profiles in the control group and Alzheimer's disease patients in Example 1 of this invention.

[0017] Figure 2 This is a technical roadmap for screening urine-specific N-glycosylated protein combinations from control populations and Alzheimer's disease patients using the urine N-glycosylation protein profiles in Example 1 of this invention.

[0018] Figure 3 This figure shows the experimental results of the Western blot method used in Example 1 of the present invention to verify the glycosylation modification of urinary N-glycosylated protein combinations in Alzheimer's disease patients and control groups.

[0019] Figure 4 This is a diagram showing the experimental results of Example 1 of the present invention, verifying the specificity and sensitivity of the Alzheimer's disease-specific N-glycosylated protein combination for diagnosing Alzheimer's disease. Detailed Implementation

[0020] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0021] Example 1

[0022] 1.1 Sample Source

[0023] The samples in this embodiment were 10 mL urine samples from control groups and Alzheimer's disease patients who had passed medical ethics review.

[0024] (1) Inclusion and exclusion criteria for the control group:

[0025] A. The inclusion criteria are as follows:

[0026] ① Age ≥ 55 years;

[0027] ②Elementary school education or above;

[0028] ③ No gender restrictions; ④ ADL (Activities of Daily Living) score ≤ 20 points;

[0029] ⑤CDR (Clinical Cognitive Impairment Scale) = 0 points;

[0030] 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.

[0031] B. Exclusion criteria include:

[0032] ① History of severe head trauma;

[0033] ② History of drug or alcohol abuse;

[0034] ③ History of mental illness such as schizophrenia or other affective disorders (including affective disorders, schizophrenia, alcohol or drug abuse or dependence, etc.);

[0035] ④ Serological tests were used to rule out low levels of Vitamin B12, low levels of folic acid, and hypothyroidism.

[0036] ⑤ No history of cognitive impairment;

[0037] ⑥ No cerebral infarction, cerebral softening, or other space-occupying lesions were found on the plain MRI scan of the head.

[0038] (2) Inclusion and exclusion criteria for Alzheimer's disease patients:

[0039] A. Inclusion criteria include:

[0040] ① Age of onset ≥ 55 years;

[0041] ②Elementary school education or above;

[0042] ③ No gender restrictions;

[0043] ④ Meets the probable diagnostic criteria for Alzheimer's disease in the International Classification of Diseases, 10th Revision (ICD-10) (for research purposes);

[0044] ⑤CDR-global≥1 point.

[0045] B. Exclusion criteria include:

[0046] ① History of severe head trauma;

[0047] ② History of drug or alcohol abuse;

[0048] ③ There are other possible causes of cognitive impairment, such as cerebrovascular diseases (including cerebral hemorrhage and cerebral infarction);

[0049] ④ History of schizophrenia or other emotional disorders;

[0050] ⑤ Serological tests should exclude low levels of Vitamin B12, hypothyroidism, and low levels of folic acid;

[0051] ⑥ The head MRI scan showed cerebral infarction, cerebral softening, and other space-occupying lesions.

[0052] Based on the MMSE score and CDR score, the severity of dementia is divided into: mild (MMSE ≥ 21 points, CDR = 1 point); moderate (MMSE 10-20 points, CDR = 2 points); and severe (MMSE ≤ 9 points, CDR = 3 points).

[0053] Table 1 below shows the basic information of the control group and Alzheimer's disease patients obtained according to the above criteria for urinary N-glycosylation protein proteomic analysis.

[0054] Table 1

[0055]

[0056]

[0057] 1.2 Evaluation Criteria

[0058] Quality control was performed using 1% Total FDR (glycopeptide FDR at spectrum level).

[0059] 1.3 Experimental Apparatus

[0060] Orbitrap Fusion TM Lumos TM Tribrid TM Thermo Scientific TM OrbitrapFusion TM Lumos TM Tribrid TM Mass spectrometer.

[0061] 1.4 Glycosylated proteomics analysis

[0062] 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.

[0063] 1.5 N-glycosylated proteomics experimental methods (specific ideas are as follows) Figure 1 (As shown)

[0064] 1.5.1 Sample Preparation

[0065] 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.

[0066] 1.5.2 Proteolysis

[0067] 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 mM TEAB, and trypsin (Promega, Madison, WI) is added at an enzyme:protein (mass ratio) of 1:50. The mixture is then 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.

[0068] 1.5.3 Enrichment of Glycosylated Peptides

[0069] 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.

[0070] 1.5.4 Nano-HPLC-MS / MS Analysis

[0071] 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 (AcclaimPepMap, 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%.

[0072] 1.5.5 Data Analysis

[0073] 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.

[0074] 1.6 By analyzing the urinary N-glycosylation proteome, specific combinations of N-glycosylation proteins in the urine of Alzheimer's disease patients were screened as candidates for subsequent diagnostic biomarkers.

[0075] Analysis of urinary N-glycosylation proteome results identified specific N-glycosylation proteins (MPO, ELANE, FGG, PON1) in the urine of Alzheimer's disease patients. These proteins were then combined as candidate diagnostic biomarkers. The specific screening and combination process is as follows: Figure 2 Firstly, this embodiment uses ProteoSpin. TM The Urine Protein Concentration MidiKit was used to extract urinary proteins from control groups and Alzheimer's disease patients. 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 proteins using virgin red as an internal control. Specific experimental results are as follows: Figure 3 As shown, for the four N-glycosylated proteins (MPO, ELANE, FGG, PON1) screened, single bands appeared in the normal population and double bands appeared in the patient population, thus correlating the glycosylation modification of the biomarker proteins with the gel running bands.

[0076] 1.7 Validation of the specificity and sensitivity of the protein combinations screened in 1.6 using urine samples from Alzheimer's disease patients and control groups.

[0077] As shown in Table 2, a sex- and age-matched normal population served as the control group. Individuals diagnosed with Alzheimer's disease (AD) were selected using a combination of clinical symptoms and Aβ / TauPET-CT (The diagnosis of dementia due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease, 2012) as the disease group (see 1.1). The specificity and sensitivity of the selected protein combinations were validated. First, N-glycosylation proteomic profiles of urine samples from the control group and AD patients were used to screen for AD-specific N-glycosylation-modified proteins as biomarkers for early diagnosis. Next, Western blot analysis was performed on a larger batch of urine samples from both groups, and the results were compared with clinical diagnoses to validate the selected biomarkers.

[0078] For the four N-glycosylated proteins (MPO, ELANE, FGG, PON1) screened: a single band in the normal population and a double band in the patient population indicate a true positive result; a double band in the normal population indicates a false positive result; and a single band in the patient population indicates a false negative result.

[0079] ROC curves were calculated to evaluate the accuracy of these four N-glycosylated proteins as biomarkers for the diagnosis of Alzheimer's disease (AD). By comparing the results and performing ROC analysis, the area under the curve was analyzed. This embodiment found that the AD-specific urinary N-glycosylated protein combination has extremely high diagnostic specificity and sensitivity. Specific results are as follows: Figure 4 As shown.

[0080] Table 2 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 (which is completely different from the control population and Alzheimer's disease patients in Table 1).

[0081] Table 2

[0082]

[0083] 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 an early diagnostic kit for Alzheimer's disease, characterized by: The urinary N-glycosylated protein combination includes N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1.

2. The application as described in claim 1, characterized in that: The urinary N-glycosylated protein group consists of N-glycosylated MPO, N-glycosylated ELANE, N-glycosylated FGG, and N-glycosylated PON1.

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