CN114280309A - Application of serum polypeptide diagnostic marker C3 for primary depression - Google Patents
Application of serum polypeptide diagnostic marker C3 for primary depression Download PDFInfo
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Abstract
The invention discloses a serum polypeptide molecule of a patient with primary depression and application thereof, wherein the amino acid sequence of a serum polypeptide molecule diagnostic marker, namely, complete Component 3 is shown in SEQ.ID.NO.1, the complete Component 3 is human Complement protein 3, and the accurate molecular weight of the complete Component 3 is 1452.12 daltons. The Component 3 presents specific high expression in the serum detection of patients with primary depression, and the expression level of the Component 3 is detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ELISA method, so that the method can be used as the detection method of the serum of patients with primary depression.
Description
Technical Field
The invention belongs to the technical field of biomarkers, and particularly relates to a serum polypeptide molecular diagnosis marker C3 for primary depression, and a detection method and application thereof.
Background
Depression is described by scientists as "cancer of the 21 st century", which is one of the main causes of global disease burden at present, and the worldwide prevalence varies from 3% to 17%, which has great influence on physical and mental health and social life of patients. Like other psychiatric disorders, the etiology of depression is extremely complex, involving psychosocial, genetic, epigenetic, neuroendocrine, and neuroimmune factors. This complexity directly affects our accuracy in diagnosing depression and its subtypes, affects our understanding of their pathophysiology, and the ability to select effective therapeutic strategies. At present, the clinical diagnosis of depression comprises medical history evaluation, mental examination and the like, the commonly used standardized patient self-rating scale and clinical scale can be used for evaluating the severity of depression symptoms of patients, the mental scale comprises PHQ-9, SDS, BDI, QIDS-SR and the like, the diagnosis strategy for early onset of depression is lacked, and the existing diagnosis method lacks sensitivity and specificity for early screening and diagnosis of depression. Therefore, the accurate molecular mechanism of the occurrence and development of the depression is discussed, and the search of a new specific diagnostic marker has important clinical significance for early diagnosis and treatment of the depression and improvement of the life quality of patients.
Before any pathological change occurs in any disease, the intracellular proteins are altered in composition and quantity and are reflected by the pattern of proteins in the serum. Therefore, by comparing the expression of different proteins in the serum of different disease populations, it is possible to screen out disease-related marker molecules. Serum proteomics refers to the study of all proteins expressed in the serum of a selected target population, and on the basis of establishing a normal Protein Expression Map (PEM), differential protein spots of the proteins are searched, and disease-related proteins are identified, so that the structure and function of the proteins are further studied, and a new way is developed for studying major disease pathophysiological mechanisms, specific markers for early diagnosis, drug action targets and the like. A large amount of proteins and polypeptides exist in human serum, and the existence, deletion and expression of partial proteins and polypeptides are closely related to the health degree of human beings, so that the human serum becomes a biomarker for disease diagnosis.
Serum diagnosis is considered to be the most recent and effective method for early diagnosis of cancer. The method judges the occurrence and development of the tumor by searching tumor markers in blood, particularly protein markers in the blood, thereby realizing early diagnosis of the tumor. A large amount of proteins and polypeptides exist in human serum, and the existence, deletion and expression of partial proteins and polypeptides are closely related to the health degree of human beings, so that the human serum becomes a biomarker for disease diagnosis.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a serum polypeptide molecule for diagnosing primary depression, a detection method and application thereof, wherein the molecular polypeptide is human Complement protein 3(C3) and is a serum marker of the primary depression.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses an application of a serum polypeptide molecular diagnostic marker C3 in preparation of a serum diagnostic reagent for primary depression, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker C3 is shown in SEQ.ID.NO. 1.
Preferably, the molecular weight of the serum polypeptide molecular diagnostic marker C3 is 1452.12 daltons.
Preferably, the serum polypeptide molecular diagnostic marker C3 has a detection parameter of 54.395-100.86pg/mL in the serum of patients with primary depression and a detection parameter of 40.865-78.54pg/mL in the serum of normal healthy people.
Preferably, the serum polypeptide molecular diagnostic marker C3 is significantly highly expressed in serum samples of patients with primary depression compared to serum samples of normal healthy people.
Preferably, the serum diagnostic marker for primary depression is a serum polypeptide molecule for ELISA detection of primary depression.
Further preferably, the serum polypeptide molecular diagnostic marker C3 is a novel target point of ELISA detection marker.
The invention also discloses application of a molecule combined with the serum polypeptide molecular diagnostic marker C3 in preparing a serum diagnostic reagent for primary depression, wherein the amino acid sequence of the serum polypeptide molecular diagnostic marker C3 is shown in SEQ ID No. 1.
The invention also discloses a diagnostic kit for primary depression, which comprises a serum polypeptide molecular diagnostic marker C3, wherein the amino acid sequence of C3 is shown in SEQ.ID.NO. 1.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a serum polypeptide molecule of primary depression, the amino acid sequence of which is shown in SEQ.ID.NO.1, and the molecule is named as supplement Component 3. The Component 3 is human Complement protein 3 with an exact molecular weight of 1452.12 daltons. The Component 3 shows specific high expression in serum detection of patients with primary depression: the range of expression in serum in normal healthy people is: 40.865-78.54 pg/mL; the range of expression in serum of patients with primary depression is: 54.395-100.86pg/mL, and there was a significant difference in expression between the two groups (p < 0.05).
Given the high specific expression of the Component 3 in the serum of the primary depression, the Component 3 can be used as a serum diagnostic marker of the primary depression; the expression level of the Component 3 is detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ELISA method, and the method can be used for detecting the primary depression patients. For the diagnosis of ELISA for detecting primary depression serum, the complete Component 3 can be used as a new target point for ELISA detection drugs.
Drawings
FIG. 1 shows the protein polypeptide peaks m/z of the present invention: 1452.12 differences in protein polypeptide expression in patients with primary depression (red) and in normal healthy population groups (green);
FIG. 2 is a gel chromatographic separation diagram of a serum protein polypeptide mixture of a primary depression patient according to the invention; wherein, the abscissa in the chromatogram represents the sample outflow time, and the ordinate represents the relative abundance of the polypeptide;
FIG. 3 is a MS/MS mass spectrometric identification profile of the compact Component 3 of the present invention; wherein, Z is 2;
FIG. 4 is the expression level of complete Component 3 protein in serum of the group of patients with primary depression and the group of normal healthy persons according to the present invention; wherein, N is 17.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The serum polypeptide molecule of primary depression provided by the invention is a newly screened serum diagnosis marker of primary depression, has specificity in expression, and can be applied to diagnosis of primary depression. The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
The specific screening method of the serum diagnosis marker for the primary depression comprises the following steps:
firstly, separating and extracting serum protein polypeptides of a primary depression patient and a normal healthy population by using a liquid protein chip technology, capturing serum protein polypeptide spectrograms of the primary depression patient and the normal healthy population by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, comparing and analyzing the difference of the serum protein polypeptide spectrograms of the primary depression patient and the normal healthy population by using ClinProTools2.1 software, finding out protein polypeptide molecules with significant differential expression among groups, and screening out a serum marker of the primary depression in a protein polypeptide peak with significant low expression in the serum of the primary depression patient.
The primary depression polypeptide serum diagnosis marker screened is verified as follows:
a protein polypeptide mixture separated from the serum of a primary depression patient is divided into 20-30 components by using HPLC (high performance liquid chromatography), secondary mass spectrum identification is carried out on the components, the identified protein polypeptide is subjected to serum regression analysis by using an enzyme-linked immunosorbent assay, and the result of serum regression verification proves that the protein polypeptide is remarkably high in expression and specificity in the serum of the primary depression patient and can be used as a biomarker for screening the serum of the primary depression patient.
1. Sample collection and processing
Samples were collected from 48 (15 men; 33 women) primary depression patients and 48 normal healthy people (18 men; 30 women) in Yanyang Hospital psychiatric department of Yanan university (9-2021-2020). The sample considers factors such as age, sex, collection time, whether the storage conditions are consistent, whether basic diseases exist and the like. Collecting blood of the collected person with fasting state, collecting 5mL whole blood with vacuum blood collection tube (yellow cap, with isolation gel), and standing at room temperature for 30 min; centrifuging at room temperature for 5min (3000g), subpackaging the upper layer serum into 100 μ L/tube, immediately storing at-80 deg.C, and avoiding repeated freeze thawing.
Reagents and instrumentation:
the serum proteins were extracted using the magnetic bead kit "weak cationic" (MB-WCX) from Bruker, Germany, as well as spectrally pure (HPLC grade) acetonitrile, trifluoroacetic acid (Merck, Germany) and alpha-cyano-4-hydroxycinnamic acid (HCCA) (Sigma, USA).
The instrument used for the experiment included a magnetic bead separator, 600/384AnchorChip target plate, and an AutoFlexIII Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS) (Bruker Daltonics, Germany).
2. Preparation of serum protein samples
A weak cation (MB-WCX) magnetic bead kit is used for capturing serum protein polypeptide, and the specific operation steps are as follows:
completely mixing the magnetic bead suspension for 1min by using a mixer;
adding 10 mu L of MB-WCX binding solution and 10 mu L of MB-WCX magnetic beads into a PCR tube, uniformly mixing, adding 5 mu L of serum, uniformly mixing for at least 5 times, and standing for 5 min;
thirdly, placing the PCR tube into a magnetic column separator, making the magnetic beads adhere to the wall for 1min, and removing the supernatant after the liquid is clear;
adding 100 mu LMB-WCX flushing fluid, moving the PCR tube on a magnetic column separator back and forth for 10 times, removing supernatant after the magnetic beads are attached to the wall, and repeating the steps three and four times;
fifthly, adding 5 mu L of MB-WCX eluent to wash the adherent magnetic beads, repeatedly blowing and beating for 10 times, allowing the magnetic beads to adhere for 2min, and transferring the supernatant into a clean centrifugal tube;
sixthly, adding 5 mu L of MB-WCX stable solution into a centrifuge tube and mixing uniformly, wherein the extracted protein polypeptide can be used for direct MALDI-TOF-MS detection or frozen in a refrigerator at the temperature of-20 ℃ for mass spectrometry within 24 h.
Mass spectrometry analysis:
mu.L of the separated and collected protein sample is mixed with 10 mu.L of matrix alpha-cyano-4-hydroxycinnamic acid, 1 mu.L of the mixture is spotted on an Anchorchip target plate (Bruker company, Germany), and three targets are spotted on each sample respectively for three times of repetition. And after drying at room temperature, putting the target plate into a mass spectrometer for flight time mass spectrometry, correcting the standard substance by adopting FlexControl 2.0 software, and then starting sample detection, wherein each sample generates a mass spectrogram after being subjected to laser targeting for 300 times (5 times of point targeting and 2 times of 30 times of targeting each time), so as to obtain protein polypeptide spectrograms consisting of different mass-to-nuclear ratios (m/z). The protein polypeptide maps of the two groups of serum samples are analyzed by using ClinProTools2.1 software, a genetic algorithm and other biological statistics and bioinformatics methods. Carrying out normalization smoothing treatment on the total ion flow diagram, and eliminating chemical and electro-physical noises; analyzing the difference protein among groups, calculating the difference size, and arranging the difference size from large to small to find out the protein polypeptide peak value (P <0.001) with obvious difference in expression among groups.
After the serum samples of the primary depression patients and the normal healthy population are processed by a magnetic bead separation system and are analyzed by MALDI-TOF-MS, protein polypeptide maps of each sample of the primary depression patients and the normal healthy population are drawn, 136 protein polypeptide peak maps are detected in the molecular weight range of 800 Da-10000 Da, and the three-time repeated stability of each sample is high.
ClinProTools2.1 software is adopted to analyze serum protein polypeptide spectra of primary depression patients and normal healthy people captured by mass spectrometry, the serum polypeptide spectra of the primary depression patients and the normal healthy people are compared and analyzed, a protein polypeptide peak map (P <0.001) with extremely obvious difference is detected, and protein polypeptides with obviously up-regulated expression in depression patients are analyzed, and the specific table is shown in Table 1.
TABLE 1 Mass Spectrometry results of peptide mapping of serum proteins for Primary Depression patients and Normal healthy people
| Molecular weight (mass to charge ratio) | P value | Mean expression of Primary Depression | Average expression of normal healthy population |
| 1452.12↑ | <0.000001 | 5.51±1.79 | 3.51±1.50 |
The results of Flex analysis software analysis of specific high-expression protein polypeptides in the serum of the primary depression patients in the table 1 are shown in the figure 1, and the results are obtained by m/z: 1452.12 in patients with primary depression (red) compared to the normal healthy population (green), m/z: 1452.12 is expressed in the serum of the patient with primary depression, so the protein polypeptide peak pattern is identified in sequence and used as the first choice of marker.
3. Sequence identification of serum potential marker of primary depression
Specifically, a technology combining liquid chromatography separation and mass spectrum is adopted to carry out the treatment on the serum polypeptide marker m/z of the patient with the primary depression: 1452.12, performing two-dimensional gel chromatography on the serum protein polypeptide remained after the mass spectrum is loaded by the magnetic bead separation and collected by adopting a nano-liter flow rate HPLC liquid phase system Easy nLC 1200 of Thermo Scientific company, and performing two-dimensional gel chromatography on the protein polypeptide m/z with the expression up-regulated in the serum of a patient with primary depression by combining an Orbitrap Fusion mass spectrometer of Thermo Scientific company: 1452.12 for sequence identification.
The specific operation steps are as follows:
3.1 sample Pre-treatment
mu.L of the sample was mixed with 800. mu.L of 5% acetonitrile 0.5% formic acid solution and added to 1.5ml of a less than 10kDa ultrafiltration tube, centrifuged at 7000 rpm for 40min in a cryocentrifuge set at 20 ℃ and concentrated to 500. mu.L. Desalting, eluting polypeptide with 70% methanol and 0.5% formic acid, collecting and concentrating to 100 μ L, and treating with Zip-tip column for concentration.
3.2 chromatographic separation
Each sample was separated using a nanoliter flow rate HPLC liquid system Easy nLC 1200. Buffer solution: the solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile solution. The column was equilibrated with 95% of solution A. The sample was applied to a mass spectrometric pre-column C18 trap column (C183 m 0.10.10X 20mm) by means of an autosampler and separated by means of an analytical column C18 column (C181.9m 0.15X 120mm) at a flow rate of 600 nl/min. The relevant liquid phase gradients are shown in table 2 and the results of gel chromatography are shown in figure 2.
TABLE 2 associated liquid phase gradients
| Time (min) | A(0.1％FA/H2O) | B(0.1％FA/ACN) | Flow rate (nL/min) |
| 0 | 92％ | 8％ | 600 |
| 8 | 88％ | 12％ | 600 |
| 48 | 77％ | 23％ | 600 |
| 68 | 64％ | 36％ | 600 |
| 69 | 5％ | 95％ | 600 |
| 78 | 5％ | 95％ | 600 |
3.3 Mass Spectrometry identification
Each sample was separated by capillary HPLC and subjected to mass spectrometry using an Orbitrap Fusion mass spectrometer (Thermo scientific). The main parameter settings are shown in table 3, and the mass spectrometric identification results are shown in fig. 3.
Table 3 main parameter settings for mass spectrometry
3.4 data analysis
Analyzing the RAW data into RAW files by mass spectrometry, performing library searching identification in a database of unidrop-human by using software sequence and a protocol discover 2.5.0(Thermo Scientific), submitting the RAW files to a sequence server through the protocol discover during library searching, selecting the well-established database, and then searching the database, wherein relevant parameters are shown in table 4:
TABLE 4 relevant parameters for database search
The result filtering parameters were: peptide FDR is less than or equal to 0.01.
The search results are shown in table 5:
TABLE 5 search results of database search
| Molecular weight | Sequence of | Position of | Protein ID | Name of protein | |
| 1452.12 | I.THRIHWESASLL.R | 1308-1319 |
| complement component | 3 |
Therefore, the method for detecting the expression level of the complete Component 3 by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an ELISA method can be used as a method for detecting patients with primary depression. Suggesting that the complete Component 3 is a protein specifically associated with primary depression, further verified by ELISA assay.
4. ELISA serum validation assay for Primary Depression serum complete 3 expression
4.1 serum samples
Serum verification analysis by ELISA was performed by collecting 17 cases of serum from patients with primary depression (8 cases in males; 9 cases in females) and 17 cases of serum from normal healthy population (8 cases in males; 9 cases in females). All serum samples were obtained from Yan Yang Hospital, Yanan university, and collected from 2020, 9 months to 2021 months.
4.2 detection method
The expression level of serum complete Component 3 of patients with primary depression and normal healthy people is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the kit is purchased from China Hengyuan biological company. The kit is used for determining the level of human complement protein 3(C3) in a specimen by using a double-antibody sandwich method. Coating a microporous plate with a purified human complement protein 3(C3) antibody to prepare a solid phase antibody, sequentially adding the complement protein 3(C3) into the microporous plate coated with the monoclonal antibody, then combining with an HRP-labeled complement protein 3(C3) antibody to form an antibody-antigen-enzyme-labeled antibody compound, and adding a substrate TMB for color development after thorough washing. TMB is converted to blue by the catalysis of HRP enzyme and to the final yellow by the action of acid. The shade of the color was positively correlated with complement protein 3(C3) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of human complement protein 3(C3) in the sample was calculated from the standard curve. The specific experimental steps refer to the kit specification, and the positive judgment standard is defined according to the kit specification.
4.3 statistical methods
The independent sample T test was performed using SPSS20 software.
4.4 analysis of results
The results of the enzyme-linked immunosorbent assay analysis show that the expression level of the Component 3 in different detection groups is that patients with primary depression > normal healthy people, and the two groups have significant difference, and the specific results are shown in table 6 and fig. 4:
TABLE 6 expression levels of complete Component 3 protein in sera of different groups
ELISA detection is carried out on the Component 3 in the serum of patients with primary depression and normal healthy people, and the result shows that the expression of the Component 3 has specificity: the range of expression in serum of patients with primary depression is: 54.395-100.86 pg/mL; the expression range in the serum of normal healthy people is as follows: 40.865-78.54pg/mL, and there was a significant difference in expression between the two groups (p < 0.05). This indicates that: the Component 3 is a protein closely related to the occurrence of the primary depression and can be used as a primary detection index of the primary depression lesion.
Therefore, the blood serum sample to be detected can be preliminarily judged to be a patient with primary depression (54.395-100.86pg/mL) or a normal healthy population (40.865-78.54pg/mL) through ELISA experiments.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<110> university of west ampere traffic
<120> application of serum polypeptide diagnostic marker C3 for primary depression
<141> 2021-11-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> Artificial Sequence
<400> 1
Ile Thr His Arg Ile His Trp Glu Ser Ala Ser Leu Leu Arg
1 5 10
Claims (8)
1. The application of the serum polypeptide molecular diagnostic marker C3 in the preparation of the serum diagnostic reagent for primary depression is characterized in that the amino acid sequence of the serum polypeptide molecular diagnostic marker C3 is shown in SEQ.ID.NO. 1.
2. The use of claim 1, wherein the molecular weight of serum polypeptide molecular diagnostic marker C3 is 1452.12 daltons.
3. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker C3 is detected in serum of patients with primary depression at 54.395-100.86pg/mL and in serum of normal healthy people at 40.865-78.54 pg/mL.
4. The use of claim 1, wherein the serum polypeptide molecular diagnostic marker C3 is significantly highly expressed in serum samples from patients with primary depression as compared to serum samples from normal healthy populations.
5. The use of claim 1, wherein the serum diagnostic marker for primary depression is a serum polypeptide molecule for ELISA detection of primary depression.
6. The use according to claim 5, wherein the serum polypeptide molecular diagnostic marker C3 is a novel target for ELISA detection markers.
7. The application of a molecule combined with a serum polypeptide molecular diagnostic marker C3 in preparing a serum diagnostic reagent for primary depression is characterized in that the amino acid sequence of the serum polypeptide molecular diagnostic marker C3 is shown in SEQ ID No. 1.
8. The diagnostic kit for the primary depression is characterized by comprising a serum polypeptide molecular diagnostic marker C3, wherein the amino acid sequence of C3 is shown in SEQ ID No. 1.
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