阅读说明：本技术 基于蛋白质组学研究抑郁或焦虑症大鼠蛋白质组变化方法 (Method for researching change of depression or anxiety rat proteome based on proteomics ) 是由 黄燕 方垂 王理想 周健 廖伟 刘燕晨 于 2020-06-20 设计创作，主要内容包括：本发明公开了一种基于蛋白质组学研究抑郁或焦虑症大鼠蛋白质组变化方法,所述方法包含下述步骤：步骤1,对大鼠大脑前额叶皮层分离与蛋白提取；步骤2,对步骤1所得的蛋白样品进行FASP法酶解、多肽iTRAQ标记、SCX分级及LC-MS/MS质谱分析；步骤3,对步骤2得到的质谱数据进行搜库与生物信息学分析；步骤4,对步骤3得到的差异蛋白进行PRM技术验证。本发明：1、利用CMS构建的临床前模型可帮助暴露抑郁症和焦虑症的潜在分子特征。2、抑郁症和焦虑症导致人类或动物前额叶皮层脑容积和树突棘减少。3、本发明采用iTRAQ定量蛋白质组学方法针对4组抑郁症/焦虑症大鼠模型的前额叶蛋白表达水平进行差异分析,提高了定量蛋白质组学结果的可靠性、客观性和准确性。(The invention discloses a method for researching change of a rat proteome of depression or anxiety based on proteomics, which comprises the following steps: step 1, separating and extracting protein from the prefrontal cortex of the brain of a rat; step 2, carrying out FASP method enzymolysis, polypeptide iTRAQ marking, SCX grading and LC-MS/MS mass spectrum analysis on the protein sample obtained in the step 1; step 3, performing library searching and bioinformatics analysis on the mass spectrum data obtained in the step 2; and 4, carrying out PRM technical verification on the differential protein obtained in the step 3. The invention comprises the following steps: 1. preclinical models constructed using CMS can help expose potential molecular features of depression and anxiety. 2. Depression and anxiety disorders result in a reduction in the volume of the human or animal prefrontal cortex brain and dendritic spines. 3. The invention adopts an iTRAQ quantitative proteomics method to carry out differential analysis on the prefrontal lobe protein expression level of 4 groups of depression/anxiety rat models, thereby improving the reliability, objectivity and accuracy of quantitative proteomics results.)

1. A proteomics-based method for studying proteome changes of rats with depression or anxiety, which is characterized in that: the method comprises the following steps:

step 1, separating and extracting protein from the prefrontal cortex of the brain of a rat;

step 2, carrying out FASP method enzymolysis, polypeptide iTRAQ marking, SCX grading and LC-MS/MS mass spectrum analysis on the protein sample obtained in the step 1;

step 3, performing library searching and bioinformatics analysis on the mass spectrum data obtained in the step 2;

and 4, carrying out PRM technical verification on the differential protein obtained in the step 3.

2. The proteomic-based method for studying changes in the proteome of rats with depression or anxiety according to claim 1, wherein the specific steps of separation and protein extraction on the prefrontal cortex of the rat brain in step 1 are as follows: taking 4 rat prefrontal lobe brain tissues of different groups respectively, weighing, homogenizing and centrifuging, and carrying out protein concentration determination by a BCA method and SDS-PAGE electrophoresis detection.

3. The proteomic-based study method of changes in the proteome of rats for depression or anxiety according to claim 2, wherein: and 2, after the protein concentration is determined in the step 1, 100ug of protein is respectively taken from each group of samples, FASP enzyme digestion is carried out, peptide fragment marking is carried out according to the specification of the iTRAQ kit, SCX high pH grading is carried out, and mass spectrum identification is further carried out.

4. The proteomic-based study method of changes in the proteome of rats for depression or anxiety according to claim 1, wherein: step 3, performing library searching on the original data acquired by the mass spectrum by using a protome distributor, and performing data filtering by using p less than or equal to 0.05; performing differential analysis on the filtered protein according to the difference multiple of more than or equal to 1.2 or less than or equal to 0.83 and p of less than or equal to 0.05, and performing biological information analysis on the obtained differential protein.

5. The proteomic-based study method of changes in the proteome of rats for depression or anxiety according to claim 1, wherein: and 4, selecting corresponding characteristic polypeptides from the differential proteins obtained in the step 3, reserving 1-3 characteristic polypeptides for each protein, and performing mass spectrum targeted quantitative analysis on the characteristic polypeptides of the differential proteins.

Technical Field

The invention relates to a method for researching change of a depression or anxiety rat proteome based on proteomics, belonging to the technical field of biomedicine.

Background

Depression and anxiety disorders are 2 common chronic neurological disorders that have negative effects on the patient's social, relatives, family and society. Many researchers find that depression and anxiety disorders have the same risk factors, including chronic irritation and stress of life. There is a lot of evidence that chronic stress life is an environmental risk factor for depression and anxiety disorders. Despite exposure to chronic stress, many individuals do not show symptoms of anxiety or depression. Chronic stress (CMS) has been widely used to induce depressive and anxiety behavior in rats to model environmental factors affecting humans. To reveal the underlying biological etiology and pathophysiology of depression and anxiety, it would be of great interest to focus on the neural substrates behind the sensitivity and resistance to stress-induced disorders.

Overall, depression and anxiety clinically present distinct core symptoms but often co-occur. Due to the common symptoms and pathogenesis that may occur, most clinical data and basic researchers are often confused, affecting our understanding of the regulatory factors for these 2 diseases. Recently, multiple researchers have slowly begun analyzing asymptomatic individuals separately from symptomless individuals to reveal unique and common features of the nervous system. Depression and anxiety disorders are heterogeneous diseases, controlled by a variety of brain structures, such as the hippocampus and the prefrontal lobe. It has been found that depression and anxiety disorders cause a reduction in the volume of the prefrontal cortex and dendritic spines in humans and animals. The prefrontal lobe is the brain area sensitive to stress, and is involved in executive, cognitive, and social emotional functions. Chronic stress-induced morphological and functional alterations of the prefrontal lobes lead to cognitive and emotional disturbances in depression and anxiety. Prefrontal plasticity abnormalities are found in both depressed and anxious individuals, and the corresponding intrinsic patterns may change fundamentally and remain unexplored to date. Therefore, there is an urgent need to identify the characteristic and common molecular characteristics of susceptibility and resistance to depression or anxiety.

In order to solve the technical problems, a new technical scheme is especially provided.

Disclosure of Invention

The present invention aims to provide a method for studying proteome changes of rats with depression or anxiety based on proteomics, so as to solve the problems presented in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a method for studying proteomic changes in rats with depression or anxiety based on proteomics, the method comprising the steps of:

step 1, separating and extracting protein from the prefrontal cortex of the brain of a rat;

step 2, carrying out FASP method enzymolysis, polypeptide iTRAQ marking, SCX grading and LC-MS/MS mass spectrum analysis on the protein sample obtained in the step 1;

step 3, performing library searching and bioinformatics analysis on the mass spectrum data obtained in the step 2;

and 4, carrying out PRM technical verification on the differential protein obtained in the step 3.

Preferably, in the step 1, the specific steps of separating the prefrontal cortex of the rat brain and extracting the protein are as follows: taking 4 rat prefrontal lobe brain tissues of different groups respectively, weighing, homogenizing and centrifuging, and carrying out protein concentration determination by a BCA method and SDS-PAGE electrophoresis detection.

Preferably, in the step 2, 100ug of protein is taken from each group of samples after the protein concentration is determined in the step 1, FASP enzyme digestion is carried out, peptide fragment labeling is carried out according to the iTRAQ kit instruction, SCX high pH classification is carried out, and mass spectrum identification is further carried out.

Preferably, the step 3 is to search the library of the raw data collected by mass spectrum by a protome discover and filter the data with p less than or equal to 0.05; performing differential analysis on the filtered protein according to the difference multiple of more than or equal to 1.2 or less than or equal to 0.83 and p of less than or equal to 0.05, and performing biological information analysis on the obtained differential protein.

Preferably, in the step 4, the corresponding characteristic polypeptides are selected from the differential proteins obtained in the step 3, 1-3 characteristic polypeptides are retained in each protein, and mass spectrum targeted quantitative analysis is performed on the characteristic polypeptides of the differential proteins.

Compared with the prior art, the invention has the beneficial effects that:

1. preclinical models constructed using CMS can help expose potential molecular features of depression and anxiety. CMS-induced depression-like behavior (happiness and behavioral despair) was assessed with SPT, FST and EMT, respectively. The established depression/anxiety rat model was divided into 4 experimental groups, a depression sensitive group, an anxiety sensitive group, a stress resistant group and a control group. By the grouping mode, depression can be distinguished from anxiety, and remarkable difference proteins related to disease sensitivity and adaptability are found through quantitative proteomics, so that the specificity and the accuracy of results are improved.

2. Depression and anxiety disorders result in a reduction in the volume of the human or animal prefrontal cortex brain and dendritic spines. The prefrontal lobe is the brain area sensitive to stress, and participates in the functions of execution, cognition, social emotion and the like. Chronic stress can induce changes in prefrontal morphology and function, leading to cognitive and emotional disturbances in depression and anxiety. The invention selects the forehead leaf of a rat model as a biological sample for quantitative proteomics analysis, and analyzes the fundamental change of protein expression profiles corresponding to depression and anxiety. Providing deeper and more targeted insight into the specific and common molecular characteristics that identify susceptibility and resistance to depression or anxiety.

3. The invention adopts an iTRAQ quantitative proteomics method to carry out differential analysis on the expression level of prefrontal lobe protein of 4 groups of depression/anxiety rat models, and carries out GO, KEGG, PPI and other analyses on the differential expression protein, thereby obtaining the biological functions, signal paths and the interaction among proteins related to different behavioral phenotype samples. And furthermore, the technology of parallel reaction detection (PRM) based on mass spectrum is adopted to carry out target verification on the found differential protein, so that the reliability, objectivity and accuracy of the quantitative proteomics result are improved.

Drawings

FIG. 1 is a flow chart of the quantitative proteomics analysis of frontal lobe tissue samples iTRAQ of the present invention.

FIG. 2 is a schematic diagram of the detection of the prefrontal lobe holoprotein quality of the present invention.

FIG. 3 is a schematic diagram of the differentially expressed proteins identified in the prefrontal lobes of the present invention.

FIG. 4 is a schematic diagram of PRM-verified differentially expressed proteins of the present invention.

Detailed Description

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.

The purpose of the invention is realized by the following technical scheme: a method for researching the proteome change of a rat with depression/anxiety neurosis based on proteomics, which analyzes the proteome change of the depression and anxiety neurosis induced by stress with high depth, high flux and high accuracy by a quantitative proteomics method, wherein the quantitative proteomics technology is iTRAQ technology and PRM technology. The method for researching the change of the proteome of the rat with depression/anxiety based on proteomics comprises the following steps:

step 1, separating and extracting protein from the prefrontal cortex of the brain of a rat;

step 2, carrying out FASP method enzymolysis, polypeptide iTRAQ marking, SCX grading and LC-MS/MS mass spectrum analysis on the protein sample obtained in the step 1;

step 3, performing library searching and bioinformatics analysis on the mass spectrum data obtained in the step 2;

and 4, carrying out PRM technical verification on the differential protein obtained in the step 3.

Preferably, in the step 1, the specific steps of separating the prefrontal cortex of the rat brain and extracting the protein are as follows: taking 4 rat prefrontal lobe brain tissues of different groups respectively, weighing, homogenizing and centrifuging, and carrying out protein concentration determination by a BCA method and SDS-PAGE electrophoresis detection.

Preferably, in the step 2, 100ug of protein is taken from each group of samples after the protein concentration is determined in the step 1, FASP enzyme digestion is carried out, peptide fragment labeling is carried out according to the iTRAQ kit instruction, SCX high pH classification is carried out, and mass spectrum identification is further carried out.

Preferably, the step 3 is to search the library of the raw data collected by mass spectrum by a protome discover and filter the data with p less than or equal to 0.05; performing differential analysis on the filtered protein according to the difference multiple of more than or equal to 1.2 or less than or equal to 0.83 and p of less than or equal to 0.05, and performing biological information analysis on the obtained differential protein.

Preferably, in the step 4, the corresponding characteristic polypeptides are selected from the differential proteins obtained in the step 3, 1-3 characteristic polypeptides are retained in each protein, and mass spectrum targeted quantitative analysis is performed on the characteristic polypeptides of the differential proteins.

Specifically, the method comprises the following steps:

as a preferred technical scheme of the invention, the detailed steps of the step 1 are as follows:

1) the rats were sacrificed by removing the neck, the head was cut along the neck, the skin of the head was cut open, and the skull was cut open to expose the brain tissue. The brain tissue was clamped out and placed in pre-chilled saline, the brain tissue containing frontal lobe tissue was cut with a blade, placed in an EP tube, and stored in a freezer at-80 ℃.

2) Taking the forehead leaf brain tissue sample out of a refrigerator at the temperature of-80 ℃, unfreezing on ice, and weighing the forehead leaf mass of each mouse. Adding lysis solution according to the ratio of the mass of the prefrontal leaves to the lysis solution of 1: 15. Homogenizing by a full-automatic rapid grinding instrument. After homogenization, the EP tubes were removed and allowed to stand on ice for 10 minutes, followed by 30 seconds of sonication per tube to further disrupt the tissue cells. Centrifuging the homogenized tissue suspension for 15 minutes at 4 ℃ at 12000 g; the supernatant was transferred to a new EP tube. Coomassie blue staining measures the concentration of the protein stock solution extracted.

3) Taking 20ug of protein sample, adding 4 Xloading buffer solution according to the volume ratio of 3:1, carrying out boiling water bath for 10 minutes, and centrifuging 12000g for 10 minutes; the supernatant was applied to 12% SDS-PAGE and electrophoresed at 80V for 90 minutes under constant pressure followed by Coomassie blue staining.

The normal saline in the step 1) is 0.9 percent of NaCl

The lysate component in step 2) is 4% SDS, 1mM DTT, 150mM Tris-HCl, pH 8.0.

The ultrasonic crushing condition in the step 3) is 70HZ for 150 s.

As a preferred technical scheme of the invention, the detailed steps of the step 2 are as follows:

1) pancreatin FASP digestion: 100ug of each protein sample was added with 200. mu.l of Buffer1, and the denatured protein was sufficiently dissolved; adding 20 mul DTT solution, reacting for 2h at 37 ℃; adding 20 μ l IAA solution, and reacting at room temperature in dark for 15 min; adding the protein solution into a 10K ultrafiltration tube, centrifuging at 12,000rpm for 10min, and discarding the solution at the bottom of the collection tube; adding 200 μ l Buffer2, centrifuging at 12,000rpm for 10min, and discarding the solution at the bottom of the collection tube; adding 200 mul TEAB Bufffer, centrifuging at 12,000rpm for 10min, and discarding the solution at the bottom of the collecting tube; replacing a new collecting pipe, adding 100 mu l of TEAB Bufffer, adding 2 mu g of mass spectrum sequencing grade trypsin (enzyme: protein is 1: 50) into each ultrafiltration pipe, and reacting for 16h at 37 ℃; centrifuging at 12,000rpm for 20min, collecting peptide fragments after enzymolysis, adding 100 μ l TEAB Bufffer into an ultrafiltration tube, centrifuging at 12,000rpm for 10min, collecting tube bottom solution, mixing with the above two solutions, centrifuging, and drying to obtain the final product.

2) iTRAQ-tagged polypeptides: polypeptide labeling is carried out according to the instruction of an iTRAQ kit of SCIEX company, all samples are mixed after labeling, and spin drying is carried out; fractionation of the peptide fragments after freeze-drying was carried out on Thermo UltiMate 3000UHPLC, column chromatography from Agilent (ZORBAX Extended-C18,2.1), detection wavelength: ultraviolet 215nm, flow rate: 0.3 ml/min; the separation gradient was a linear rise of mobile phase B from 5% to 38% in 80 min. Collect 1 tube every 1 minute in gradient range, collect 16 tubes of elution solution, centrifuge dry for LCMS analysis.

3) LC-MS/MS analysis: one-dimensional chromatographed polypeptide samples were dried by centrifugation, redissolved in Nano-LC mobile phase a (0.1% formic acid), bottled, and analyzed by on-line LCMS. The solubilized sample was loaded onto a nanobipe C18 pre-column in a volume of 2 μ L (3 μm,) And then 20ul volume elution for desalting, the liquid phase is Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), the sample is desalted and retained on the pre-column, and then separated by analytical column, the specification of the analytical column is C18 reversed phase chromatographic column (50 μm × 15cm C18-2 μm)) The gradient used in the experiment was an increase of mobile phase B (80% acetonitrile, 0.1% formic acid) from 8% to 38% within 50 min. The mass spectrum adopts a ThermoFisher Q active system (ThermoFisher, USA) combined with a Nano-liter spraying Nano Flex ion source (ThermoFisher, USA), the spraying voltage is 1.9kV, the heating temperature is 275 ℃, the mass spectrum scanning mode is under an information-Dependent acquisition working mode (DDA, Data Dependent Analysis), the scanning time of a single spectrum of a primary MS is 100MS, at most 14 secondary spectrums with the charge of 2+ to 4+ are acquired under each DDA cycle, and the accumulation time of each secondary spectrum is 50 MS. Each cycle time was fixed at 1.8 seconds, the collision cell energy setting was adapted for all precursor ion collision induced dissociation (HCD), and the dynamic exclusion setting was 25 s.

The Buffer1 in the step 1) is 8M urea/100mM Tris-HCl, pH 8.5;

the Buffer2 in the step 1) is 8M urea/100mM Tris-HCl, pH 8.0;

the mobile phase 2 in the step 2) is 10mM ammonium formate, 90% acetonitrile and pH10.0;

as a preferred technical scheme of the invention, the detailed steps of the step 3 are as follows:

1) library searching analysis and quantification: processing the obtained mass spectrum original spectra of each component by using a protome scanner software (v2.1.0.81) to perform database retrieval and protein identification and relative quantitative analysis, wherein the PSM false positive rate FDR is set to be 1%, and the rest database retrieval parameters are set as follows: and (3) labeling and quantifying the iTRAQ 8-plex peptide fragment, wherein the database is a Rattusnorvegicus protein database, the mass error of the pancreatin enzyme digestion and primary mass spectrum is 10ppm, and the mass error of the secondary mass spectrum is 0.05 Da. Performing identification quality evaluation on qualitative and quantitative results of the polypeptides and the proteins obtained by searching the library, and performing t-test statistical analysis on the quantitative results; and defining the protein with the difference multiple more than or equal to 1.2 times and less than or equal to 0.83 times and the statistical test p value less than or equal to 0.05 as the differential protein, thereby obtaining the change condition of the rat prefrontal lobe proteome expression under different stress conditions.

2) Bioinformatics analysis: performing GO function annotation (http:// www.geneontology.org) on the differential protein obtained in the step 1); and further performing Pathway analysis on the KEGG database to determine the main physiological and biochemical metabolic pathways and signal regulation pathways involved by the protein.

As a preferred technical scheme of the invention, step 4 is to carry out PRM verification on part of differential proteins obtained in step 3, and the detailed steps are as follows:

1) and (3) obtaining a biological sample by reference to the step 1 and the step 2, and performing enzyme digestion to obtain enzyme digestion polypeptide. Taking the database searching result obtained in the step 3 and the original data as database building data, and importing the database building data into Skyline software for library building; and screening a target polypeptide list of the differential protein from the DDA result, and performing PRM analysis by using the target polypeptide list as a quantitative target polypeptide of the differential protein.

2) PRM validation of the peptide fragment of interest: after the enzyme-cleaved polypeptide sample is centrifugally dried, the enzyme-cleaved polypeptide sample is re-dissolved in a Nano-LC mobile phase A (0.1% formic acid), bottled and loaded, and subjected to online LCMS analysis. The solubilized sample was loaded onto a nanobipe C18 pre-column in a volume of 2 μ L (3 μm,) Then desalted by 20ul volume flush. The liquid phase was Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), and the sample was desalted on a pre-columnSeparating with analytical column (50 μm × 15cm C18-2 μm) of C18 reverse phase chromatography) The gradient used in the experiment was an increase of mobile phase B (80% acetonitrile, 0.1% formic acid) from 8% to 38% within 90 min. Mass Spectrometry A ThermoFisher Q active system (ThermoFisher, USA) was used in combination with a nanoliter spray Nano Flex ion source (ThermoFisher, USA) at a spray voltage of 1.9kV and a heating temperature of 275 ℃. The main scanning parameters include: MS2 resolution 30000, isolation window (1.2 m/z), AGC set to 5e5, maximum integration time 200MS, HCD energy 28.

3) Importing the off-line data verified by the PRM obtained in the step 2) into Skyline software, standardizing the quantitative data by the Skyline software, checking the peak shape of the target peptide segment, and judging the spectrogram effect. Quantitative information of the target peptide fragments is derived, and the quantitative value of the protein is calculated in a peptide fragment adding mode and is used for statistical analysis among groups.