Application of blood protein markers in the preparation of products for diagnosing mild cognitive impairment

Abstract

The present invention provides an application of a protein marker in blood in the preparation of a product for diagnosing mild cognitive impairment, and relates to the field of biotechnology. The present invention provides an application of a protein marker in blood in the preparation of a product for diagnosing mild cognitive impairment, wherein the protein marker includes at least one of MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3‑35. The inventors have found that compared with patients without mild cognitive impairment, the above 8 proteins are differentially expressed in the blood of patients with mild cognitive impairment, and have the potential to serve as diagnostic markers for mild cognitive impairment. Further studies have found that using the above 8 proteins together as markers can effectively distinguish mild cognitive impairment from normal elderly controls, which greatly facilitates clinical transformation applications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of a protein marker in blood in preparing a product for diagnosing mild cognitive impairment. Background Art

[0002] With the increasing aging of the population, the high prevalence of Alzheimer's disease (AD) has become a serious public health problem. AD ​​is currently difficult to diagnose early and there is a lack of effective therapeutic drugs. The academic community believes that an important reason for the failure of many AD drug developments is the lack of identification and intervention in the early stages, so early diagnosis of AD is extremely important. Mild cognitive impairment (MCI) is an early stage in the progression of AD. If it can be identified and intervened at this stage, it will help prevent or delay the further development of MCI into AD. Currently, the diagnosis of AD mainly relies on cerebrospinal fluid and imaging methods, which have disadvantages such as high cost and invasiveness. Patient compliance in clinical practice is poor, and the development of blood test indicators will help overcome these problems. Previous proteomics studies reported that the combination of MCI or AD blood diagnostic markers usually requires the detection of dozens of proteins, which is not conducive to clinical translation.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The first object of the present invention is to provide an application of a protein marker in blood in the preparation of a product for diagnosing mild cognitive impairment, so as to solve at least one of the above problems.

[0005] The second object of the present invention is to provide a product for diagnosing mild cognitive impairment.

[0006] In a first aspect, the present invention provides a use of a protein marker in blood in the preparation of a product for diagnosing mild cognitive impairment;

[0007] The protein markers include at least one of MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35.

[0008] As a further technical solution, the protein markers are MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35.

[0009] As a further technical solution, the protein marker is differentially expressed in samples of patients with mild cognitive impairment compared with those of patients without mild cognitive impairment.

[0010] As a further technical solution, the expression of MMP9 is reduced in samples of patients with mild cognitive impairment compared to patients without mild cognitive impairment;

[0011] SERPINA11 expression is elevated in samples from patients with mild cognitive impairment;

[0012] PI16 expression is elevated in samples from patients with mild cognitive impairment;

[0013] CRTAC1 expression is elevated in samples from patients with mild cognitive impairment;

[0014] MMRN1 expression is elevated in samples from patients with mild cognitive impairment;

[0015] C4B expression is elevated in samples from patients with mild cognitive impairment;

[0016] C1RL expression is elevated in samples from patients with mild cognitive impairment;

[0017] IGHV3-35 expression was decreased in samples from patients with mild cognitive impairment.

[0018] As a further technical solution, the non-mild cognitive impairment patients include at least one of normal elderly people, Huntington's disease patients, frontotemporal dementia patients and amyotrophic lateral sclerosis patients.

[0019] As a further technical solution, the sample includes blood.

[0020] In a second aspect, the present invention provides a product for diagnosing mild cognitive impairment, wherein the product is used for detecting the expression level of the protein marker.

[0021] As a further technical solution, the product includes a marker for identifying the protein marker.

[0022] As a further technical solution, the marker includes an antibody that binds to the protein marker.

[0023] As a further technical solution, the product includes a reagent or a kit.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The inventors have found that compared with patients with non-mild cognitive impairment (normal elderly people, Huntington's disease patients, frontotemporal dementia patients and amyotrophic lateral sclerosis patients), MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35 are differentially expressed in the blood of patients with mild cognitive impairment, and have the potential to serve as diagnostic markers for mild cognitive impairment. The inventors have further found that the marker composed of the above 8 proteins can efficiently distinguish mild cognitive impairment from normal elderly controls (AUC = 0.881). Compared with the MCI or AD blood protein diagnostic markers reported in existing proteomics studies, which usually require the detection of dozens of protein molecules, the MCI blood diagnostic protein molecule marker provided by the present invention has high sensitivity and specificity (AUC = 0.881), and reduces the number of protein molecules that need to be detected to 8, which greatly facilitates clinical translation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The proteomics analysis process provided in Example 1;

[0028] Figure 2 The targeted proteomics analysis process provided in Example 2;

[0029] Figure 3 The protein markers and test results provided in Example 3. DETAILED DESCRIPTION

[0030] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0031] In a first aspect, the present invention provides a use of a protein marker in blood in the preparation of a product for diagnosing mild cognitive impairment;

[0032] The protein markers include at least one of MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35.

[0033] The inventors have found that the above protein markers are differentially expressed in the blood of patients with mild cognitive impairment compared with patients without mild cognitive impairment (normal elderly people, patients with Huntington's disease, patients with frontotemporal dementia and patients with amyotrophic lateral sclerosis).

[0034] Specifically, MMP9 expression was reduced in blood samples of patients with mild cognitive impairment compared with patients without mild cognitive impairment;

[0035] SERPINA11 expression is elevated in samples from patients with mild cognitive impairment;

[0036] PI16 expression is elevated in samples from patients with mild cognitive impairment;

[0037] CRTAC1 expression is elevated in samples from patients with mild cognitive impairment;

[0038] MMRN1 expression is elevated in samples from patients with mild cognitive impairment;

[0039] C4B expression is elevated in samples from patients with mild cognitive impairment;

[0040] C1RL expression is elevated in samples from patients with mild cognitive impairment;

[0041] IGHV3-35 expression was decreased in samples from patients with mild cognitive impairment.

[0042] Therefore, the eight protein markers provided by the present invention have the potential to serve as diagnostic markers for mild cognitive impairment.

[0043] Furthermore, the protein markers are MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35.

[0044] Further research by the inventors revealed that the eight-protein marker described above can effectively differentiate between mild cognitive impairment and normal elderly controls (AUC = 0.886). Compared to existing proteomics studies reporting blood protein diagnostic markers for MCI or AD, which typically require the detection of dozens of protein molecules, the MCI blood diagnostic protein marker provided by the present invention reduces the number of detected protein molecules to just eight, while maintaining comparable diagnostic performance, significantly facilitating clinical translational applications.

[0045] In a second aspect, the present invention provides a product for diagnosing mild cognitive impairment, wherein the product is used for detecting the expression level of the protein marker.

[0046] The product provided by the present invention realizes the diagnosis of mild cognitive impairment by detecting the expression levels of protein markers (MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35) in samples.

[0047] In some preferred embodiments, the product includes a marker that identifies the protein marker.

[0048] The identification and quantification of protein markers are achieved through markers.

[0049] In some preferred embodiments, the label comprises an antibody that binds to the protein marker.

[0050] In some preferred embodiments, the product comprises a reagent or a kit.

[0051] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0052] Example 1 TMT Whole Proteomic Analysis of Serum Samples from AD Patients and Control Groups

[0053] Proteomic analysis was performed on 105 serum samples (13 cases of severe Alzheimer's disease, 12 cases of moderate Alzheimer's disease, 6 cases of mild Alzheimer's disease, 7 cases of mild cognitive impairment, 20 cases of Huntington's disease, 20 cases of amyotrophic lateral sclerosis, 20 cases of normal elderly controls, and 7 biological replicates (one sample was randomly selected for each type of repeated testing)). Figure 1 shown).

[0054] The specific proteomic analysis methods are as follows:

[0055] 1) 4 μL of serum was treated with 175 μL of a high-abundance protein removal resin (Thermo Scientific, A36372) according to the manufacturer's instructions. The protein was then denatured in 6 M urea / 2 M thiourea in a 3 kDa protein concentrator tube and concentrated to 50 μL. The protein lysate was then reduced and alkylated by the addition of 10 mM tris(2-carboxyethyl)phosphine (TCEP) and 40 mM iodoacetamide (IAA) and incubated at 32°C in the dark for 40 minutes. After further dilution with 100 mM ammonium bicarbonate, the protein was digested with 0.5 μg of intracellular protease at 32°C for 4 hours, followed by 0.625 μg of trypsin at 32°C for 12 hours. The reaction was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 1%. The trypsin-digested peptides were then cleaned and desalted using a desalting column, and the peptides were labeled using TMTpro16plex according to the manufacturer's instructions.

[0056] 2) Nanoliquid chromatography-high-resolution mass spectrometry

[0057] The re-dissolved serum peptides were analyzed by LC-MS / MS. Our experiment was designed in batches, and samples of different diseases were randomly assigned to different batches to minimize the impact of batch effects on our proteomics data. We randomly divided 105 samples into 7 batches for TMTpro 16plex labeling, with the same number of samples in each batch (such as Figure 1 For each batch of TMT samples, a DIONEX UltiMate 3000RSLCnano system (ThermoFisher Scientific, San Jose, USA) and an XBridge Peptide BEH C18 column ( 5μm×4.6mm×250mm)(Waters, Milford, MA, USA) for separation. The sample was separated by gradient separation in 10mM ammonia water (pH=10.0) using 5% to 35% acetonitrile (ACN) (i.e., the proportion of acetonitrile gradually increased from 5% to 35%) at a flow rate of 1mL / min. The TMT-labeled peptides were separated into 60 fractions by this system, and these fractions were further combined into 30 fractions. After spin drying, these 30 fractions were redissolved with 2% ACN / 0.1% formic acid (FA), and the redissolved peptides were analyzed by LC-MS / MS. Data for each fraction was collected using the nanoflow DIONEX UltiMate 3000RSLCnano system (Thermo Fisher Scientific, San Jose, USA) in combination with a QE-HF high-resolution mass spectrometer (Thermo Fisher Scientific, San Jose, USA) using data-dependent acquisition (DDA) mode. During analysis, the sample was first loaded onto a precolumn (3 μm, The sample loaded on the pre-column was then flushed onto an analytical column (1.9 μm, 120 Å, 150 mm x 75 μm) at a flow rate of 300 nL / min for further separation. The separation time was 60 minutes, and the LC gradient was from 5% to 28% buffer B (buffer B was 98% ACN (containing 0.1% FA), and buffer A was 2% ACN, 98% H2O (containing 0.1% FA)). All reagents were MS grade. MS parameters included an m / z range of 350 to 1800, a resolution of 60,000 (at 200 m / z), an AGC of 3e6, and a maximum ion injection time (max IT) of 50 ms. The first 15 precursor ions were selected for secondary fragmentation in MS / MS with a resolution of 45,000 (200 m / z), an AGC of 2e5, and a maximum IT of 120 ms. The isolation window for the selected precursors was 0.7 m / z. Mass spectrometric data were analyzed using Proteome Discoverer (version 2.4.1.15, Thermo Fisher Scientific) and the protein database (downloaded from UniProtKB). The enzyme was set to trypsin with two missing cleavage tolerances. Static modifications were carbamidomethylation of cysteine ​​(+57.021464) and TMTpro at the N-terminus of lysine residues and peptides (+304.207145). Variable modifications were oxidation of methionine (+15.994915) and acetylation at the N-terminus of the peptide (+42.010565). The precursor ion mass tolerance was set to 10 ppm, and the fragment ion mass tolerance was set to 0.02 Da.

[0058] The results of the whole proteomic analysis are as follows:

[0059] A total of 1,702 proteins were detected in the serum samples. A differential analysis of the serum proteins was then performed to identify differentially expressed proteins between patients with mild cognitive impairment, Huntington's disease, and amyotrophic lateral sclerosis (ALS) compared with normal elderly controls. Comparative analysis of these differentially expressed proteins ultimately identified 37 differentially expressed proteins unique to mild cognitive impairment, indicating that these 37 differentially expressed proteins are potential diagnostic markers for mild cognitive impairment.

[0060] Example 2 Validation of differentially expressed proteins in an independent validation cohort

[0061] Targeted proteomics testing (e.g., Figure 2 shown).

[0062] The specific proteomic analysis methods are as follows:

[0063] The proteolysis method and whole proteomics preparation method were the same as in Example 1, except that the removal of high-abundance proteins, TMT labeling, and fractionation were not performed. Parallel reaction monitoring (PRM) experiments were performed using a nanoflow DIONEX UltiMate 3000RSLCnano system (ThermoFisher Scientific) with a QE-HF hybrid quadrupole-orbitrap ion trap (Thermo Fisher Scientific). 0.5 μg of peptide sample was injected for each PRM experiment. The flow rate was 300 nL / min (pre-column 3 μm, Inner diameter 20mm*75μm; analytical column 1.9μm, Serum peptides were separated using a flow rate of 150 mm (150 mm x 75 μm) with an effective gradient of 30 min (buffer B increasing from 10% to 30%). Buffers A and B were consistent with those used in the whole proteomics experiment. A total of 52 peptides were included in the serum PRM experiment, including 13 internal standard peptides (CiRT). The resolution of the primary MS and PRM was 60,000 (m / z 200) and 30,000 (m / z 200), respectively. The AGC target was set to 2e5, and the maximum time interval (IT) for PRM was set to 80 ms. PRM data were analyzed using Skyline. Retention times were predicted using the CiRT peptides, and the separation time window was set to 5 min. The primary MS and MS / MS mass analyzers were set to "Orbitrap" with resolutions of 60,000 and 30,000, respectively. Protein quantification data were converted to peptide quantification data using ProteomeExpert.

[0064] The results of targeted proteomics analysis are as follows:

[0065] The 12 potential differentially expressed proteins in Example 1 that can be used for MCI diagnosis were verified in serum.

[0066] Example 3 Classification of MCI and Normal Elderly Control Samples

[0067] We used machine learning methods at the proteomic level to evaluate the differences between the above-obtained proteins in MCI and normal elderly control samples, and to find protein markers for classification.

[0068] The data mining process is as follows:

[0069] Machine learning was performed using the R package randomForest (version 4.6.14) with modifications briefly described below. Key random forest parameters, including the cutoff value, mean reduced accuracy, number of cross-validation iterations, and number of trees, were optimized. Input protein features were selected based on the mean reduced accuracy cutoff. Five cross-validations were performed. Serum models were constructed for 600 strains. The minimum reduced mean accuracy for serum protein features was set to 0. The mtry value for the serum model was set to the square root of 2.

[0070] We explored the potential for distinguishing MCI from normal elderly samples based on molecular signatures of dysregulated proteins. We constructed a random forest machine learning model based on serum proteomic data from 23 MCI patients and 45 control participants. We selected a panel of eight core proteins to distinguish MCI from control samples ( Figure 3 The model was tested using a cohort of 8 MCI and 21 CN patients, with an AUC of 0.881 ( Figure 3 (shown in B).

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of reagents for detecting protein markers in blood in the preparation of products for diagnosing mild cognitive impairment; The protein markers are a combination of MMP9, SERPINA11, PI16, CRTAC1, MMRN1, C4B, C1RL and IGHV3-35.

2. The use according to claim 1, characterized in that Compared with non-mild cognitive impairment patients, the protein markers are differentially expressed in samples of patients with mild cognitive impairment.

3. The use according to claim 2, characterized in that Compared with patients without mild cognitive impairment, the expression of MMP9 is reduced in samples from patients with mild cognitive impairment; SERPINA11 expression is elevated in samples from patients with mild cognitive impairment; PI16 expression is elevated in samples from patients with mild cognitive impairment; CRTAC1 expression is elevated in samples from patients with mild cognitive impairment; MMRN1 expression is elevated in samples from patients with mild cognitive impairment; C4B expression is elevated in samples from patients with mild cognitive impairment; C1RL expression is elevated in samples from patients with mild cognitive impairment; IGHV3-35 expression was decreased in samples from patients with mild cognitive impairment.

4. The use according to claim 2, characterized in that The non-mild cognitive impairment patients include at least one of normal elderly people, Huntington's disease patients, frontotemporal dementia patients and amyotrophic lateral sclerosis patients.

5. The use according to claim 2, characterized in that The sample comprises blood.

6. A product for diagnosing mild cognitive impairment, characterized in that: The product is used for detecting the expression level of the protein marker described in claim 1; The product comprises a marker that recognizes the protein marker of claim 1; The label includes an antibody that binds to the protein marker.

7. The product according to claim 6, characterized in that The product includes a reagent or a kit.

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