CN116879564A - Spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology and application thereof - Google Patents
Spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology and application thereof Download PDFInfo
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- CN116879564A CN116879564A CN202311143906.4A CN202311143906A CN116879564A CN 116879564 A CN116879564 A CN 116879564A CN 202311143906 A CN202311143906 A CN 202311143906A CN 116879564 A CN116879564 A CN 116879564A
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
Abstract
The invention provides a spinal cord injury biomarker based on proteomics and phosphorylated protein modification genetics, which is selected from one or more of the following substances: SYN1, SYN1 Ser62. Through the research of proteomics and phosphorylation protein modification groups of spinal cord injury, SYN1 and SYN1 Ser62 with obvious difference in expression level in spinal cord injury are finally screened out and used as biomarkers, the application of the biomarkers in the aspects of spinal cord injury diagnosis and prognosis is provided initially, and a new target point is provided for researching medicines for treating spinal cord injury.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a spinal cord injury biomarker based on proteomics and phosphorylated protein modification group and application thereof.
Background
Spinal cord injury (Spinal cord injury, SCI) is the most serious complication of spinal injuries. Various causes cause damage to spinal cord structural function, resulting in various motor, sensory and sphincter dysfunctions, dystonia, and corresponding changes in pathological reflexes, etc., at the corresponding segment of the damage. Spinal cord injuries can be classified into traumatic spinal cord injuries and non-traumatic spinal cord injuries according to the causative factors: traumatic spinal cord injury is mainly caused by external factors, and non-traumatic spinal cord injury is often caused by tumor compression, vascular ischemia or congenital diseases. Spinal cord injury is classified into primary injury and secondary injury according to pathophysiological mechanisms: the primary injury of spinal cord refers to the initial mechanical injury caused by the local deformation of spinal column, and the direct compression and injury of bone fragments or intervertebral disc materials which are fractured and shifted after mechanical injury to neurons and blood vessels; secondary mechanisms are triggered by primary injury, secondary mechanisms include a range of biochemical and cellular processes such as electrolyte abnormalities, free radical formation, vascular ischemia, edema, post-traumatic inflammatory responses, apoptosis, or gene-programmed cell death.
Currently, treatments for spinal cord injuries mainly include: (1) Drug therapy, mainly comprising large-dose hormone impact, gangliosides, minocycline, cethrin (Rho pathway inhibitor), and the like. (2) The early operation decompression treatment of the spinal cord injury achieves better effect in the aspect of reducing nerve injury, the operation decompression treatment not only relieves the compression on the injured spinal cord, but also plays a positive role in stabilizing the plastic spinal column, and provides a stable environment for spinal cord recovery. (3) Hemodynamic treatment, preventing hypotension after spinal cord injury, and even increasing mean arterial pressure, may be beneficial for neurological recovery. (4) Stem cell therapies, mainly including neural stem cells, hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, and the like. (5) Gene therapy and tissue engineering, there is increasing evidence that biological and engineering strategies have shown therapeutic potential in spinal cord injury. While the effectiveness of these treatment options provides modest benefits to spinal cord injured patients, the injury inflicted on the patient is not removed at all. Thus, the search for new targets for the treatment of spinal cord injuries is an urgent need for spinal surgery today.
Proteomics is the science of studying the composition of proteins and their activity rules in cells, tissues or whole life at the whole level, using proteins as subjects. Protein characteristics, including protein expression levels, post-translational modifications, protein-protein interactions, etc., are studied by proteomics, thus obtaining comprehensive insight on the protein level regarding the processes of disease occurrence and disease progression, etc. In proteomics, the key technology for identifying proteins in biological samples is Mass Spectrometry (MS). Since the spinal cord injury occurrence mechanism is a complex and intricate process involving a number of factors, the use of proteomics can help people to understand the pathogenesis and treatment of spinal cord injury more deeply.
With the rapid development of phosphorylated protein modification groups, phosphorylated protein modification groups have been applied to the fields of biology and medicine. The main application is as follows: (1) disease occurrence and treatment study: phosphorylated protein modification histology can help identify and verify protein and phosphorylation targets related to diseases, and can provide basis for diagnosis, treatment and drug development of diseases. (2) cell Signal transduction study: phosphorylated protein modification histology may reveal the role of proteins in cell signaling, helping us understand the complexity and dynamics of cell signaling networks. (3) protein interaction study: phosphorylation modifications can affect the structure and function of proteins, revealing the characteristics and function of the protein interaction network. (4) drug development and evaluation: in the development of drugs, phosphorylated protein modification histology techniques can be used to identify and evaluate the targets and mechanisms of action of the drugs; in terms of drug evaluation, phosphorylated protein modification histology may be used to assess the effect of a drug on the phosphorylation state of a protein.
Synpsin-1 (SYN 1) is called Synapsin 1, which is one of the most widely studied members of the Synpsin family, and is mainly expressed in the presynaptic membrane and synaptic vesicles of the central nervous system, and is an important regulator of presynaptic membrane transport and release of synaptic vesicles. In addition, SYN1 has great potential in phosphorylation control mechanism, and is close to the occurrence and development of nervous system diseases. However, SYN1 is still in the preliminary stage of the spinal cord injury study, and the role of phosphorylation regulation of SYN1 on spinal cord injury is not yet clear.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology and application thereof, which can be used for early diagnosis and prevention of spinal cord injury, biological protein targeted therapy, prognosis monitoring judgment and the like.
One of the objects of the present invention is a spinal cord injury biomarker based on proteomics and phosphorylated protein modification group, selected from one or more of the following: SYN1, SYN1 Ser62.
Another object of the invention is the use of a spinal cord injury biomarker based on proteomics and phosphorylated protein modification group for the preparation of a product for diagnosing spinal cord injury, said biomarker being selected from one or more of the following: SYN1, SYN1 Ser62.
Further, the product is a detection reagent, a kit, a microarray or a biochip.
Further, the biomarkers are useful for detection of samples from spinal cord tissue by analytical means of proteomics and phosphorylated protein modification group.
Another object of the invention is the use of a spinal cord injury biomarker based on proteomics and phosphorylated protein modification group in the manufacture of a medicament for treating spinal cord injury, said medicament targeting said biomarker, said biomarker being selected from one or more of the following: SYN1, SYN1 Ser62.
Through the research of proteomics and phosphorylation protein modification groups of spinal cord injury, SYN1 and SYN1 Ser62 with obvious difference in expression level in spinal cord injury are finally screened out and used as biomarkers, the application of the biomarkers in the aspects of spinal cord injury diagnosis and prognosis is provided initially, and a new target point is provided for researching medicines for treating spinal cord injury.
Drawings
FIG. 1A is a full proteome data box diagram of example 1 of the present invention;
FIG. 1B is a full proteome PCA plot of example 1 of the present invention;
FIG. 2A is a graph of a linear fit of the mean (In (mean)) to the standard deviation (In (sd)) of the full protein abundance of example 1 of the present invention;
FIG. 2B is a histogram of the residual error of embodiment 1 of the present invention;
FIG. 2C is a contour plot of rank versus residual for the full protein abundance average of example 1 of the present invention;
FIG. 2D is a Q-Q plot of the residual error of example 1 of the present invention;
FIG. 3A is a volcanic plot of DEPs of example 1 of the present invention, with red labeled up-regulated protein, gray labeled no-differential change protein, and blue labeled down-regulated protein;
FIG. 3B is a cluster thermal graph analysis of DEPs of example 1 of the present invention, blue for up-regulated protein and cyan for down-regulated protein;
FIG. 4A is a graph of the bubble pattern of the whole proteome differential protein GO assay of example 1 of the present invention;
FIG. 4B is a bar graph of the whole proteomic difference protein KEGG analysis of example 1 of the present invention;
FIG. 4C is a graph of whole proteome GSEA enrichment analysis of example 1 of the present invention;
FIG. 5A is a statistical map of phosphorylation data of example 1 of the present invention;
FIG. 5B is a graph showing the ratio of the phosphorylation data in example 1 of the present invention;
FIG. 5C is a statistical map of phosphorylated proteins of example 1 of the present invention;
FIG. 5D is a statistical chart of phosphorylated peptide fragments according to example 1 of the present invention;
FIG. 5E is a graph of the phosphorylation site duty cycle circle of example 1 of the present invention;
FIG. 6A is a PCA plot of whole proteome data of example 1 of the present invention;
FIG. 6B is a PCA plot of phosphorylated protein modification set data of example 1 of the present invention;
FIG. 7A is a graph of a linear fit of the mean (In (mean)) to the standard deviation (In (sd)) of phosphorylated protein abundance of example 1 of the present invention;
FIG. 7B is a histogram of the residual error of embodiment 1 of the present invention;
FIG. 7C is a volcanic diagram of a modified group of phosphorylated proteins according to example 1 of the present invention;
FIG. 7D is a map of the differential site of the modified group of phosphorylated proteins of example 1 of the present invention;
FIG. 8A is a graph of a linear fit of the mean (In (mean)) to the standard deviation (In (sd)) of the phosphorylation rate of example 1 of the present invention;
FIG. 8B is a histogram of the residual error of embodiment 1 of the present invention;
FIG. 8C is a volcanic plot of the phosphorylation rate group according to example 1 of the present invention;
FIG. 8D is a map of the differential sites of the phosphorylation rate group according to example 1 of the present invention;
FIG. 9 is a Wen diagram of the phosphorylation difference site of the modified group of phosphorylated proteins in example 1 of the present invention;
FIG. 10A is a graph of the GO enriched bubbles of the protein to which the differential phosphorylation site of example 1 belongs according to the present invention;
FIG. 10B is a GO chord chart showing the biological process of the protein to which the differential phosphorylation site of example 1 of the present invention belongs;
FIG. 10C is a thermal map of synaptic tissue-related protein expression according to example 1 of the present invention;
FIG. 10D is a schematic diagram of the synaptic tissue key protein TOP 10 according to example 1 of the present invention;
FIG. 10E is a bar graph of the protein KEGG enrichment to which the differential phosphorylation sites of example 1 belong;
FIG. 11A is a kinase-substrate network diagram of spinal cord injured synaptic tissue of example 1 of the present invention, kinase on the left and substrate on the right;
FIG. 11B is a kinase-substrate network diagram of spinal cord injury SYN1 of example 1 of the invention, with non-kinase-substrate linkage on the horizontal line, kinase-substrate linkage on the vertical line (P > 0.05), and kinase-substrate linkage on the orange square (P < 0.05);
FIG. 12A is a SYN1 highly correlated protein GO enrichment assay of example 1 of the invention;
FIG. 12B is a SYN1 highly correlated protein KEGG enrichment analysis of example 1 of the invention;
FIG. 13A is SYN1 whole proteome statistics of example 1 of the present invention;
FIG. 13B is SYN1 phosphorylated protein modification group statistics of example 1 of the present invention;
FIG. 14A is a full protein level SYN1 Western Blot of example 1 of the present invention;
FIG. 14B is a Western blot of phosphorylated protein levels SYN1 Ser62 of example 1 of the present invention.
Detailed Description
The design, positive sample verification and result analysis of the present application are further described below in conjunction with specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Example 1
1 Experimental materials and instruments
1.1 animal origin
All experiments used C57BL/6 mice (purchased from the university of medical science laboratory animal institute in south China) of SPF grade for adult females, aged 6-8 weeks, body weight 17-23 g.
1.2 Experimental major reagents
BCA protein quantification kit (Thermo); western and IP cell lysate (Meilunitio); protease inhibitor cocktail (PI) (Roche); phosphatase Inhibitor (PPI) (Roche); fe-mac phosphorylation enrichment column (Sep-Pak); gold water (mass spectrometry grade) (Thermo Fisher Science); formic Acid (FA) (mass spectrometry grade) (Sigma-Aldrich); trifluoroacetic acid (TFA) (Sigma-Aldrich); acetonitrile ACN (mass spectrometry grade) (Thermo Fisher Science); anhydrous methanol (guangzhou chemical reagent plant); iodoacetamide (IAA) (Sigma-Aldrich); dithiothreitol (DTT) (Sigma-Aldrich); UREA (UREA) (Sigma-Aldrich); trypsin (raw Xia Danbai V5280); absolute ethanol (guangzhou chemical reagent plant); tetraethylammonium bromide (TEAB) (Sigma-Aldrich); iRT Kit (Biognosys); tween (Tween) (Sigma-Aldrich); ECL chemiluminescent substrate luminophore (Bio-Rad); PVDF membrane (Millipore); 5X Loading buffer (Bio-Rad); protein Marker (Bio-Rad); skim milk powder (Genebase); ammonium Persulfate (APS) (Sigma-Aldrich); sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich); tetramethyl ethylenediamine (TEMED) (Sigma-Aldrich); acrylamide (Sigma-Aldrich); 1.5M Tris-HCl buffer (Sigma-Aldrich).
1.3 experiment of primary antibodies
Rabbit anti-SYN 1 monoclonal antibody (1:1000) (Proteintech); rabbit anti-SYN 1 Ser62 monoclonal antibody (1:500) (Bioworid); beta-action (I102) polyclonal antibody (1:2000) (Bioworid); rabbit secondary antibody (1:5000) (Bioworid).
1.4 Main experiment instrument
Ice making machine (Hubei Huang Danshi medical equipment factory); electronic balance instruments (Orhaus instruments Co.); cryogenic tissue mill (Shanghai He Fan instruments Co., ltd.); non-contact ultrasonic instruments (Xinzhi biology company); refrigerated centrifuge (Hunan instrument); mass spectrometer (Lumos Obitrap); microplate reader (Biotek); a vacuum desalination pump (Agilent); millopore pure water system (guangzhou royal instrument technology limited); constant temperature culture shaking table (Shanghai-constant group); water bath (Changzhou common instrument manufacturing Co., ltd.); vortex finders (linbell instruments, inc., seadoor); freeze dryer (Hunan Hexi instruments Co., ltd.); protein gel electrophoresis apparatus (Bio-Rad); full-automatic chemiluminescence image analysis system (Shanghai tenable).
1.4 preparation of Main reagents
(1) Mass spectrum experiment related reagent formula
a) 8M Urea:24.024 Dissolving Urea in 50 mL gold water, and adjusting pH to 8.0 with concentrated hydrochloric acid
b) 1M DTT:154.2 mg of DTT is dissolved in 1 mL golden water
c) 1M IAA:184.96 Dissolving IAA in 1 mL golden water
d) Conditioning Buffer:0.1% TFA in 20% acetonitrile solution
e) Washing Buffer:0.1% TFA in 5% acetonitrile solution
f) Elution Buffer:0.1% TFA in 60% acetonitrile solution
(2) Western Blot related reagent formula
a) 5% concentrated gel
Ultrapure water 2.1. 2.1 mL
30% monomer 0.5. 0.5 mL
1.5M Tris-HCl pH 6.8 0.38 mL
10% SDS 30 μL
10%APS 30 μL
TEMED 3 μL
b) 10% separating gel
Ultrapure water 6.9. 6.9 mL
30% monomer 4 mL
1.5M Tris-HCl pH 8.8 0.75 mL
10% SDS 60 μL
10% APS 60 μL
TEMED 9 μL
c) Running buffer (10×)
Tris-base 30.3 g
Glycine (Glycine) 144.4 g
SDS 10 g
Pure water 1000 mL
d) Transfer film buffer solution (10×)
Tris-base 37.9 g
Glycine (Glycine) 187.7. 187.7 g
Pure water 1000 mL
e) Transfer film buffer solution (1×)
10 Xtransfer buffer 80 mL
Anhydrous methanol 200 mL
Pure water 720 mL
f) TBS buffer (10×)
Sodium chloride 80 g
Potassium chloride 2 g
Tris-base 30 g
Pure water 1000 mL
g) TBST buffer (1×)
10 XTBS buffer 50 mL
Tween 20 500 μL
450 mu L of pure water
h) 5% skimmed milk
Skimmed milk powder 2.5 g
1 XTBST buffer 50L
2 Experimental methods
2.1 Establishing a spinal cord injury model of a mouse
(1) This example uses C57BL/6 mice of SPF grade from adult females of 17-23 g, 6-8 weeks of age, to construct a spinal cord injury model. The 6 mice were randomly divided into 2 groups: sham surgery group (Sham) and Injury group (Injury). The 2 groups of mice were fasted prior to anesthesia and were deeply anesthetized with tribromoethanol by intraperitoneal injection. After the anesthesia is successful, the back is shaved, the back is placed on an operating table in a prone position, and conventional iodophor is used for disinfection.
(2) The injured mice were used as experimental groups to make skin incisions of 2 cm length starting from the highest point of the spine, and the back skin muscles of the mice were blunt-isolated to expose the T9-T11 spinal segments. And laminectomy centered on T10, fully exposing the spinal cord. The LISA spinal cord impactor is used for injury, the computer parameters are adjusted to be 1.8 mm in depth, 0.35 s in time and 1.25N in average impact force for impact, so that rapid swelling and congestion of spinal cord tissues can be seen, tail swing reflection occurs at the tail, and the two lower limbs flutter. After successful molding, subcutaneous tissue and skin are sutured sequentially.
(3) Sham mice served as a control group, and were subjected to skin incision alone, without treatment of spinal cord, and subcutaneous tissue and skin were sutured sequentially.
(4) The mice were kept warm at 37 ℃ after surgery and waited for anesthesia and recovery. Mice were kept in separate cages to ensure adequate food source. After successful molding, all mice were subcutaneously injected with 1. 1 mL of 0.9% NaCl solution containing antibiotics to prevent infection, and artificial bladder massage and urination was performed daily for 3 d.
2.2 Mouse spinal cord tissue harvesting
(1) The 6 mice are deeply anesthetized by intraperitoneal injection with pentobarbital sodium, and after the anesthesia is good, the mice are prone to be placed on an operation table for conventional iodophor disinfection.
(2) Skin was dissected from the back skin of the mice with a scalpel, the muscles were blunt-separated, and the spinal column of the mice was exposed to perform laminectomy, exposing the entire length of spinal cord covered by dura mater.
(3) The full length of the spinal cord of the mice was extracted, spinal dura mater was removed, rinsed 3 times with PBS, and placed in 2 mL cryopreservation tubes, respectively, and rapidly frozen with liquid nitrogen, and stored in a-80 ℃ refrigerator.
2.3 Tissue milling and lysing
(1) The spinal cord tissue of the mice is taken out in a refrigerator at the temperature of minus 80 ℃ and placed on dry ice, the spinal cord tissue is sheared in an ultra clean bench, and the crushed spinal cord tissue is respectively placed in a 2 mL grinding tube. The grinding beads are cleaned by absolute ethyl alcohol and are sucked by absorbent paper, 3 small grinding beads are added into each grinding tube, and grinding is carried out by a precooled tissue grinding instrument (working condition: 70 Hz,2 min, -50 ℃) until the grinding is carried out to powder.
(2) To the tissue milling tube was added PPI and PI-containing (Roche, 10X and 50X after dissolution with 1 mL gold water, respectively) cell lysate 1 mL. Taking out the grinding beads in the tissue grinding tube by using forceps, pre-cooling to 4 ℃ by using non-contact ultrasonic, starting a pre-cooling system for water circulation, and performing ultrasonic crushing (10 s at intervals of 10 minutes in ultrasonic 5 s at 4 ℃ and 10 minutes continuously) on the tissues in the grinding tube, so that the tissue particles are completely cracked.
(3) The tissue-containing grind tube was placed on ice for 30 min for lysis with 1 shaking vigorously every 10 min. The lysed tissue was centrifuged in a pre-chilled centrifuge (working conditions: 4 ℃, 12000 rpm, 30 min), and the supernatant was pipetted with a pipette and transferred to a 1.5 mL centrifuge tube.
2.4 High efficiency proteolysis
(1) Detecting protein concentration of the spinal protein sample liquid extracted in the previous step through a BCA kit: the protein standard was diluted with ultrapure water in a gradient to prepare standard samples having final concentrations of 0. Mu.g/. Mu.L, 0.0625. Mu.g/. Mu.L, 0.125. Mu.g/. Mu.L, 0.25. Mu.g/. Mu.L, 0.5. Mu.g/. Mu.L, 1. Mu.g/. Mu.L, and 2. Mu.g/. Mu.L, respectively. BCA working solutions were prepared in a ratio of 200 μ L B to 4 μ L A per sample, incubated in an incubator at 37 ℃ for 30 minutes, and absorbance at 570 nm was read using a microplate reader. According to the protein concentration detected by the BCA kit, taking 3 mg protein sample liquid with corresponding volume, and placing the protein sample liquid into a 15 mL centrifuge tube for enzymolysis.
(2) Adding 8M Urea with proper volume into a 15 mL centrifuge tube to make the final concentration of the sample larger than 4M (namely 1:1), taking the sample with the lowest concentration as the standard, and filling the rest samples with higher concentration into the same volume by using the 8M Urea, and fully vibrating.
(3) Preparing DTT (gold water preparation) of 1M, adding a proper amount of volume to ensure that the working concentration is 50 mM (namely 1:20), fully vibrating, and carrying out water bath 1 h at 37 ℃ to fully reduce disulfide bonds of protein.
(4) IAA (prepared by using golden water) of 1M is prepared, and a proper volume is added to ensure that the working concentration is 135 mM, and the IAA is fully vibrated and placed at room temperature in a dark place for 30 min.
(5) The ultrafiltration tube is rinsed with 1 mL of TEAB, and is centrifuged (working condition: 20 ℃, 4000 rpm, 20 min), if the liquid is not completely removed, the centrifugal angle is required to be changed to 90 degrees, so that the membrane of the ultrafiltration tube is prevented from being damaged until the liquid is completely removed, and the membrane of the ultrafiltration tube cannot be prevented from being damaged by centrifugation for more than 15 min each time.
(6) And adding the sample into the rinsed ultrafiltration tube, centrifuging (working condition: 20 ℃ C., 4000 rpm, 20 min) until the volume of each tube is not more than 1 mL, and if the liquid is not completely removed, switching the centrifugal angle to 90 degrees to avoid the breakage of the ultrafiltration tube membrane and centrifuging until the liquid is completely removed, wherein the centrifugal time is not more than 15 min, and the breakage of the ultrafiltration tube membrane is avoided.
(7) 1 mL of 8M Urea was added to the ultrafiltration tube, shaken well, centrifuged until the liquid was completely removed (working condition: 4 ℃, 4000 rpm, 20 min), and repeated 2 times.
(8) 1 mL of TEAB was added to the ultrafiltration tube, and the mixture was thoroughly shaken and centrifuged (working condition: 4 ℃ C., 4000 rpm, 20 min) until the liquid was completely removed, and repeated 5 times. The collection tube is discarded and replaced with a new collection tube.
(9) A company-matched HCL solution was used to dissolve pancreatin to prepare a pancreatin concentration of 1. Mu.g/. Mu.L, and 50 mM TEAB 500. Mu.L and 75. Mu.L of the prepared mass-spectrum-grade pancreatin solution were sequentially added to the ultrafiltration tube.
(10) Blowing off flocculent protein in an ultrafiltration tube with a clean gun head to make the solution turbid, sufficiently shaking for 5 min, sealing the ultrafiltration tube with a preservative film, shaking the ultrafiltration tube with a shaking table at 200 rpm at 37 ℃ for 16-18 h, blowing off flocculent protein again with the clean gun head after 8 h to make the solution turbid, and sufficiently shaking for 5 min.
(11) Centrifuging the ultrafiltration tube in the last step to collect the tube (working condition: 4 ℃, 4000 rpm, 20 min), adding 1 mL gold water, blowing off flocculent protein by a clean gun head to make the solution turbid, sufficiently shaking for 5 min, centrifuging (working condition: 4 ℃, 4000 rpm, 20 min), and collecting peptide solution after enzymolysis.
(12) And (5) detecting the concentration of the peptide fragment solution subjected to enzymolysis by using the BCA kit, and calculating the enzymolysis efficiency of the protein sample.
2.5 High flux desalination
(1) And (5) connecting a vacuum desalting instrument, and detecting tightness. And cleaning the desalting column connector by pure water.
(2) Activating and desalting column: the Sep-pak C18 desalting column was activated with methanol for 10 min, and the vacuum pump pressure was controlled to not exceed 300 kpa.
(3) Balance desalting column: 2% acetonitrile containing 0.1% FA was prepared as a equilibration solution (Condition Buffer) and passed through the column 2 times, each time 1. 1 mL, for 1 min.
(4) Sucking a sample: placing a waste liquid pipe on a frame of the negative pressure tank, allowing the peptide solution to pass through the column for 3-5 times, 1 mL each time, and allowing the peptide solution to pass through the column for 1 min for the first time, and desalting by a vacuum pump to avoid air bubbles generated at too high flow speed due to too large pressure difference, thereby leaking liquid.
(5) Cleaning a sample: gold water containing 0.1% of FA was used as a Washing solution (Washing Buffer), and the column was repeated 5 times for 1. 1 mL each time.
(6) Eluting the sample: the collection tubes were placed on the shelves of the negative pressure tank, and first, 200. Mu.L each of the collection tubes were passed through the column 3 times using 40% acetonitrile (Elution Buffer) containing 0.1% FA.
(7) Again, 80% acetonitrile with 0.1% FA was used as eluent (solution Buffer), 200. Mu.L each time, 3 times over the column. 200 mu L of peptide fragment sample liquid is taken as the whole protein mass spectrum detection, and freeze-dried and desalted peptide fragment sample liquid is used by a cold trap and a vacuum pump. And freeze-drying the desalted peptide fragment sample liquid of the residual sample by using a cold trap and a vacuum pump.
2.6 Enrichment of phosphorylated peptide fragments
(1) The Binding/Wash Buffer in the phosphorylation enrichment kit is pre-adjusted with FA to adjust the pH value to be lower than 3. 200 mu L Binding/Wash Buffer was used to dissolve the lyophilized peptide, vortex shaker was used to shake, and after dissolution, the pH of the dissolved peptide was ensured to be below 3.
(2) The protective cap at the bottom of the centrifuge column was carefully removed, and the screw cap was loosened but not removed. The column was placed in a 2 mL waste tube and centrifuged (working condition: 4 ℃, 1000rpm, 30 s) to remove the preservation solution. The nuts are removed and stored for preparation for subsequent steps.
(3) 200. Mu.L of Binding/Wash Buffer was applied to the column. 1000 g, centrifuging 30 and s, and discarding the waste liquid. This step was repeated 1 time. The bottom of the column was plugged with a stopper and transferred to a new centrifuge tube.
(4) 200. Mu.L of the eluate (solution Buffer) from the phosphorylation enrichment kit was used to dissolve the peptide into the equilibrated column and the screw cap was tightened. The screw cap was grasped and the bottom stopper was gently tapped until the resin was completely dissolved (no vortex or inversion of the post was done to prevent the resin from splashing onto the post inner wall). Incubating for 30 min, and repeating the step (2) 1 time every 10 min.
(5) The screw cap and bottom plug were removed slightly (without squeezing the plug to reflux the liquid into the column). The column was transferred to a centrifuge tube and centrifuged (working conditions: 4 ℃, 1000rpm, 30 s). 200. Mu.L of Binding/Wash Buffer was added to the column and centrifuged (working conditions: 4 ℃, 1000rpm, 30 s) and repeated 2 times. 200. Mu.L of gold water was added to the column and centrifuged (working conditions: 4 ℃, 1000rpm, 30 s). The column was transferred to a new centrifuge tube.
(6) 100. Mu.L of the solution Buffer was added to the column, centrifuged (working condition: 4 ℃, 1000 rpm, 30 s) and repeated 1 time (the liquid became brown, which is a normal phenomenon, if the amount of peptide fragment was less than 1 mg, 0.1% formic acid was added for Elution). The phosphorylated peptide fragment samples were lyophilized using a lyophilizer.
2.7 Liquid chromatography and mass spectrometry combined analysis (LC-MS/MS)
(1) The lyophilized whole protein sample after desalting was dissolved with 20. Mu.L of 0.1% FA (prepared with gold water), the concentration was measured with BCA kit, and the amount of 0.1% FA to be added was adjusted according to the measured concentration to give a final concentration of 0.5. Mu.g/. Mu.L.
(2) Removing particles: each sample was centrifuged at 15. Mu.L to a fresh EP tube (working conditions: 4 ℃, 12000g, 20 min). The supernatant was centrifuged in a fresh tube (working condition: 4 ℃ C., 12000g, 20 min). 9.5. Mu.L of the supernatant was taken into a new centrifuge tube, 0.5. Mu.L of the target peptide (Irt) was added, vortexed and mixed well, and centrifuged (working conditions: 4 ℃, 12000g, 10 min).
(3) Each sample was taken in 5. Mu.L to a new coupon. Note that the coupon cannot have bubbles.
(4) Each sample after the above described degranulation was taken 3 μl to the same centrifuge tube and mixed well, and 15 μl was aspirated into the coupon (carefully creating air bubbles) for DDA banking. DDA was injected into 3 needles, each 5. Mu.L.
(5) The lyophilized phosphorylated peptide fragment sample is dissolved in 0.1% FA (gold water), and the phosphorylated peptide fragment sample is added into a sample loading tube after removing particles according to the above steps.
(6) And (3) placing the upper sample tube into a mass spectrum detection instrument, respectively adopting a DDA mode detection sequence and a DIA mode detection sequence for mass spectrum analysis by the phosphorylated protein modification group, and adopting a DIA model for mass spectrum analysis by the whole protein group.
2.8 Searching a database
In the embodiment, the qualitative analysis of the DDA data is performed by using a Maxquat platform to establish a mouse phosphorylated protein database, and the quantitative analysis of the DIA data is performed by using Spectronaut software. And respectively leading out map data comparison results of the whole protein group and the phosphorylase group after comparison with a mouse protein database.
2.9 Statistical analysis method for bioinformatics
2.9.1 Analysis using software
The bioinformatics analysis was performed mainly using R software (4.2.0). The present embodiment considers that the missing value processing on the data is a conservative method: an excessively large deviation of the quantitative protein value (i.e., below 500) is defined as a missing value, and the quantitative protein missing value is average-filled.
2.9.2 Sample reproducibility evaluation
In this embodiment, biological repeated samples are taken during the extraction of the protein expression amount, so that the protein repeatability between samples is evaluated by adopting principal component analysis (Principal Component Analysis, PCA) and Box-plot (Box-plot) visualization, so as to check whether the experimental results of the biological repeated samples have statistical consistency, and the accuracy and the reliability of the experiment are improved.
2.9.3 Whole proteome differential analysis
Based on quality control of whole proteome data, the prior research shows that a power law global analysis (PLGEM) model is favorable for carrying out statistical analysis on the proteome data, and is also one of proteome analysis models commonly used by the team. The present example fits the whole proteome quantitative results by PLGEM model and evaluates the data quality. Differential analysis was performed using PLGEM model, and volcanic and heat map visual display was performed on differential proteins based on R language using ggplot2 package.
2.9.4 protein functional enrichment assay
This example functionally annotates the differential protein and sets an enrichment test significance P value of less than 0.05.
GO (Gene ontologies) enrichment analysis reveals the biological functions and interrelationships of different genes and Gene sets by classifying and annotating genes, mainly from three levels: molecular function, cellular components and biological processes functionally analyze proteins or genes.
KEGG (Kyoto encyclopedia of genes and genomes, encyclopedia of kyoto genes and genome) is a systematic biological database integrating information on genome, biochemical reactions, metabolic pathways, etc. Enrichment analysis of differential proteins can help researchers understand the functions and interactions of genes and proteins in metabolic pathways, providing important clues for studying biological processes, discovering new biomarkers, and developing drugs.
Gene set enrichment analysis (Gene Set Enrichment Analysis, GSEA): the method is used for analyzing the enrichment condition of the gene set in the high-flux gene expression data under different experimental conditions so as to study the relationship between the gene expression change and the biological process. In this example, whole proteome data was subjected to GSEA to preliminarily determine the effect of synergistic changes in genes within the gene set on phenotypic changes.
2.9.5 Phosphorylated protein modification group and phospho change rate differential analysis
Based on the above data quality control, in this embodiment, the phosphorylation change rate is calculated by using the phosphorylated protein modification group and the whole white matter group (that is, the abundance of the phosphorylated peptide is divided by the abundance of the corresponding protein), and the difference analysis is performed on the data results of the phosphorylated protein modification group and the phosphorylated change rate group by using the PLGEM model, and the difference phosphorylation site overlapping the same direction change in the two groups is found as the difference phosphorylation site.
2.9.6 Functional enrichment analysis of proteins to which differential phosphorylation sites belong
Functional enrichment analysis is carried out by combining the screened phosphorylated protein differential sites with a GO database and a KEGG database, and the functions and interactions of the protein related to the differential phosphorylated sites in metabolic pathways participated in after spinal cord are discussed.
2.9.7 Kinase-substrate regulatory network construction
To explore the mechanisms involved in the regulation of phosphorylation of spinal cord injury and the associated effects of phosphorylated kinases, iGPS software is a highly developed version based on GPS that predicts proteins of kinases potentially regulated by the site of phosphorylation modification. Kinase prediction is carried out on the differential phosphorylation sites through iGPS software, a kinase-substrate regulation network after spinal cord injury is constructed, and a kinase-substrate network diagram is drawn by using Cystonemap.
2.9.8 Functional enrichment analysis of SYN1 highly correlated proteins
Based on whole proteome data, a correlation analysis of SYN1 was performed, proteins with a correlation coefficient >0.7 were defined as SYN1 highly correlated proteins, and GO functional enrichment analysis and KEGG enrichment analysis were performed.
2.10 Western blot (immunoblotting experiment)
(1) Preparing a sample: protein sample concentrations were detected using BCA kit, and each sample was taken an equivalent amount of protein (about 30-100 μg as required) to a new EP tube, and the volume was filled with cell lysate, added with 5 x Loading buffer, heated in 95 ℃ water bath for 10 min, and cooled on ice.
(2) Preparing electrophoresis gel: cleaning 1.5. 1.5 mm thin glass plate and thick glass plate with clear water, airing at normal temperature, and clamping and fixing by a frame. The tightness of the glue making rack was checked with pure water. Firstly adding 10% of separating gel, adding absolute ethyl alcohol to drive off bubbles, flattening the separating gel, after waiting for 20-30 min, solidifying the separating gel, adding 5% of concentrated gel, horizontally inserting comb teeth, and after waiting for 20-30 min, solidifying the concentrated gel.
(3) Loading: clamping a glass plate by using an electrophoresis tank, filling the prepared electrophoresis liquid into the inside and the outside, horizontally taking out comb teeth, avoiding loading Kong Waixie, and adding a sample into a loading hole by using a pipetting gun;
(4) Electrophoresis: and (3) inserting a power supply, and adjusting parameters: constant pressure 80V. After the sample runs through the gel, the voltage is adjusted to 120V, and electrophoresis is stopped according to the molecular weight of the target protein and the molecular weight of the reference protein, so that the sample is prevented from running away from the gel;
(5) Transferring: placing pre-cooled 1L membrane transferring liquid into an operation panel, then placing a sandwich clamp into the membrane transferring liquid, placing a sponge and filter paper on the black surface, shearing a small piece of PVDF membrane of 8×5 cm by scissors, cutting the angle at the upper right corner, so that the reverse direction after membrane transferring can be avoided, peeling off a thin glass plate after electrophoresis is finished, cutting concentrated glue, reserving separating glue, lightly separating the separating glue, lightly placing the separating glue on the filter paper, then soaking the prepared PVDF membrane in methanol for 1 min to achieve the aim of activation, then placing the PVDF membrane on the separating glue, adjusting the position, enabling the PVDF membrane to be fully covered on the separating glue, and simultaneously avoiding bubbles generated under the PVDF membrane to influence the membrane transferring. The whole process needs to be gentle, and separation gel rupture is avoided. And (3) placing the sandwich clamp into an electric rotating groove, pouring the film transferring liquid in the operating panel into the electric rotating groove, ensuring that the PVDF film is fully soaked in the electric rotating liquid, and placing 1 ice box at the rest empty positions so as to ensure the safety of electric rotation. Adjusting parameters: constant voltage 100V, current 235 mA, 120 min beginning to turn;
(6) Closing: preparing 5% sealing liquid, putting the PVDF film after film transfer into a small box containing TBST (containing Tween), cleaning for 2 times, removing TBST (containing Tween), adding the prepared 5% sealing liquid, taking the sealing liquid as a reference, and slowly shaking on a shaking table for 1 h;
(7) Incubating primary antibodies: after the sealing is finished, removing sealing liquid, adding TBST (containing Tween) to clean the PVDF film, putting the PVDF film on a shaking table to shake for 5 min, removing TBST (containing Tween), repeating the cleaning step for 3 times to achieve the aim of removing all sealing liquid, soaking the PVDF film in the prepared primary antibody solution, and then putting the PVDF film on the shaking table at 4 ℃ to shake slowly overnight;
(8) Incubating a secondary antibody: recovering primary antibody solution, adding TBST (containing Tween) to wash PVDF membrane, shaking for 5 min, removing TBST (containing Tween), repeating the washing step for 3 times, adding prepared secondary antibody, soaking PVDF membrane in the secondary antibody solution, and shaking slowly for 1 h;
(9) Developing: removing the secondary antibody solution, adding TBST (containing Tween) to clean the PVDF film, shaking for 5 min, removing TBST (containing Tween), repeating the cleaning step for 3 times, taking the prepared luminous solution, tweezers, paper towel and PVDF film to the biomolecule imager, slightly clamping the PVDF film by using tweezers, putting the PVDF film into the paper towel with the front side facing upwards, sucking residual TBST (containing Tween) as much as possible, putting the residual TBST into the biomolecule imager, dripping the luminous solution on a target protein strip for development, and preserving the result.
3. Experimental results and statistical analysis
P <0.05& Foldchange >1.5 was considered as a statistically different change, and all data analyses were performed in R4.2.0 software.
3.1 quality control of Whole proteome Mass Spectrometry data
Since proteome data quality affects subsequent analysis and validation, quality control of proteome data is required, and proteome data quality is assessed by data distribution comparison and PCA analysis in this example. The quality control results shown in FIGS. 1A and 1B indicate that the protein expression level distribution trend of 6 samples is similar. However, the protein expression level was different in the spinal cord injury group compared with the control group (sham operation). The difference between the two groups is smaller and the difference between the groups is larger. The result shows that the overall data has small systematic error and is suitable for further exploration.
3.2 Whole proteome differential analysis
The PLGEM model is commonly used to fit genome and proteome expression datasets. The PLGEM model was therefore used to fit the whole proteome data of spinal cord injury, followed by screening for differentially expressed proteins of statistical significance. The results show that the average value of the full protein abundance data and the standard deviation are in a linear relation, r2 is 0.993 (figure 2A), the rank of the residual abundance average value is unbiased, and the residual abundance average value belongs to normal distribution (figures 2B, 2C and 2D), so that the protein group data has good linear fitting degree on the protein group data for repairing spinal cord injury, and can be used for subsequent analysis.
This example defines the protein of PLGEM model differential analysis P <0.05 and Foldchange >1.5 as differential protein (spinal cord injury group vs control; different Proteins, DEPs). In the whole proteome data 472 differential proteins were selected, including 348 up-regulated differential proteins and 124 down-regulated differential proteins (fig. 3A). The heat map fully demonstrates that the screened DEPs can clearly distinguish between the control group and the spinal cord injury group (FIG. 3B).
3.3 Total proteome differential protein enrichment analysis
To further explore the biological processes and cellular functions affected by spinal cord injury, GO enrichment analysis was performed on 472 differential proteins (fig. 4A). From the results, it can be seen that the biological processes (Biological Process, BP) are concentrated in small molecule catabolism, positive regulation of external stimulus response, negative regulation of phosphorylation, response to oxidative stress, synaptic tissue, dephosphorylation, and neuronal death related biological processes. Wherein synaptic tissue is a specific structure of neural signaling between neurons. Following spinal cord injury, the structure and function of neurons and synapses are altered, which may lead to poor or abnormal signaling between neurons, further affecting the normal function of the nervous system. The pathway of oxidative stress is also one of the important signaling pathways of spinal cord injury, affecting neuronal function, and is associated with spinal cord injury treatment and prognosis. However, the regulatory mechanisms of phosphorylation can regulate biological processes such as neuronal metabolism, and movement. The cell fraction (Cellular Component, CC) is mainly enriched with collagen-containing extracellular matrix, synaptic membrane, mitochondrial matrix, endoplasmic reticulum membrane, etc., and the molecular function (Molecular Function, MF) is mainly enriched with oxidoreductase, lyase activity, phosphatase activity, phosphoester hydrolase activity, etc. In the regulatory mechanisms of phosphorylation, phosphorylases and phosphatases can regulate excitability and repression of spinal cord neurons, thereby affecting the release behavior and neurotransmitter release of neurons.
In addition, KEGG enrichment analysis results for the differential proteins suggested significant aggregation of glycine, serine, threonine metabolism and sphingomyelin metabolic pathways (fig. 4B), directly reflecting the local metabolic profile following spinal cord injury. GSEA enrichment was performed on all proteins, and the results suggest that amino acid metabolism, carboxylic acid metabolism, organic acid metabolism, small molecule metabolism, steroid metabolism, etc. were mainly enriched (fig. 4C). Previous researches show that metabolic abnormality is one of common complications after spinal cord injury, metabolism is an indispensable factor in spinal cord injury, and the metabolic change of an organism after spinal cord injury leads to the increase of incidence rate of dyslipidemia, diabetes, cardiovascular diseases and the like, and is closely related to the progress and prognosis of the diseases.
In conclusion, the enrichment analysis result of the differential protein shows that the pathophysiological mechanism of the spinal cord injury is particularly complex, the treatment and prognosis of the spinal cord injury are affected, however, the phosphorylation regulation and control enriches the disease mechanism of the spinal cord injury, and the deep exploration of the phosphorylation regulation and control of the spinal cord injury has important significance.
3.4 spinal cord injury phosphorylated protein modification histology statistics
As shown in FIGS. 5A-5E, the data of the spinal cord injury phosphorylated protein modification group show that 2730 phosphorylated proteins and 11265 phosphorylated peptide fragments are totally identified, and the ratio of the phosphorylated proteins to the phosphorylated peptide fragments is 89.6 percent and 77.9 percent respectively, which shows that the phosphorylated enrichment effect is good. Of the 11265 phosphorylated peptides, there were a total of 5274 phosphorylation sites, with the proportion of phosphorylation at serine (Ser), threonine (Thr) and tyrosine (Tyr) being 90.36%, 8.84%, 0.80%, respectively, similar to other mammalian phosphorylated protein modification groups.
3.5 quality control of phosphorylated protein modification group Mass Spectrometry data
The phosphorylated protein modification group evaluates the data quality of the protein group through data distribution comparison and PCA analysis, as shown in quality control results of fig. 6A and 6B, the distribution of total protein expression amounts of 6 samples is similar, the intra-group difference is small, the inter-group difference is obvious, the overall systematic error of the phosphorylated protein modification group data is small, the repeatability of the prosthetic operation group and the spinal cord injury group is good, and the inter-group difference is large, so that the method is suitable for subsequent analysis.
3.6 phosphorylated protein modification group and phosphorylated Change Rate group PLGEM analysis
For analysis of phosphorylated protein modification panel data, if a comparison of differences in the abundance of phosphorylated proteins between panels is made directly, the change in such differences cannot be accurately determined as being from the protein itself or from a change in the level of phosphorylation. Thus, this example uses the full protein abundance to correct for phosphorylated peptide fragment abundance, calculates the rate of change in phosphorylation (i.e., the abundance of phosphorylated peptide fragment divided by the abundance of the corresponding protein), and then uses the PLGEM model to perform differential analysis on the phosphorylated protein modification group and the phosphorylated rate of change group, respectively.
Phosphorylated protein modification group data showed r=0.994 (Pearson r=0.843) at PLGEM fitting results (fig. 7A), and residual values were consistent with normal distribution (fig. 7B). The phosphorylated protein modification group was screened for 590 differential sites by differential analysis (P <0.05& foldchange > 1.5), including 314 up-regulation sites and 276 down-regulation sites (fig. 7C and 7D).
The phosphorylation rate group showed r=0.994 at PLGEM fitting results (fig. 8A), and the residual values were in line with normal distribution (fig. 8B). The phosphorylation rate panel was screened for 641 differential sites, including 341 up-regulation sites and 300 down-regulation sites, by differential analysis (P <0.05& foldchange > 1.5) (fig. 8C and 8D).
3.7 differential phosphorylated protein site screening
To improve the reliability of the data, this example defines overlapping sites with the same directional differential variation between the phosphorylated protein modification group and the phosphorylated rate of change group as differential phosphorylated sites. The results indicated that the differential phosphorylation sites of this example totaled 434 differential sites, including 245 up-regulation sites and 189 down-regulation sites (fig. 9).
3.8 protein enrichment analysis to which differential phosphorylation sites belong
To further investigate the function of the protein to which the differential phosphorylation site belongs, GO enrichment analysis was performed on the protein to which 434 differential phosphorylation sites belong. The results show that the biological process is mainly concentrated in the paths of synaptic tissues, axon formation, dendritic development, protein autophosphorylation and the like. Enriching synaptic organization, neurotransmitter transport, neurotransmitter secretion, modulation of neuronal synapses, etc. in cellular localization; actin binding, phospholipid binding, tubulin binding, cytoskeletal structure, etc. are enriched in molecular function (fig. 10A). Where neurotransmitter transmission, vesicle localization and synaptic organization are all important components of neural signaling, critical to the normal functioning of the nervous system, and SYN1 is the only intersection of the four aforementioned neural signaling pathways (fig. 10B). In the spinal cord, neurons communicate through synapses, whose structure and function are regulated by neurotransmitter and vesicle localization. Protein changes on the synaptic tissue signal pathway help to understand the changes in the signal transduction pathway following spinal cord injury, and mass spectrometry results indicate that the changes in the pathway for different proteins following spinal cord injury are distinct (fig. 10C). Further analysis using the synapse-associated proteins in conjunction with the String database, a key protein for TOP 10 on the pathway was found, indicating that SYN1 is the central protein on the pathway (FIG. 10D). Furthermore, the KEGG database was mainly enriched for glycometabolism, axonal guidance, erbB signaling pathways, carbon metabolism, and synaptic vesicle cycling, etc. (fig. 10E).
In summary, protein enrichment analysis of the phosphorylated differential sites reflects local features of spinal cord injury and identifies signaling pathways following spinal cord injury. Synaptic tissue is one of the key pathways through which spinal cord injury neurons transmit signals, and the most significantly altered protein on this pathway is SYN1.
3.9 Kinase-substrate network construction
In this example, kinase predictive analysis was performed on the differential phosphorylation sites by iGPS software to obtain a kinase-substrate interaction table after spinal cord injury. By kinase-substrate network construction of the phosphorylation sites of synapse-associated proteins (FIG. 11A), the results indicate that kinases have a regulatory effect on the proteins SYN1, MARCKS, GAP43, GPHN, RPH 3. Alpha. Wherein the multiple phosphorylation sites of SYN1 are downstream substrates, ser62, ser67, ser510, ser551, ser553, ser605 (fig. 11A), respectively, we constructed a spinal cord injury SYN1 kinase-substrate regulatory network. The results show that upstream kinases of SYN1 mainly comprise CAMK2 alpha, CAMK2G, CDK, CDK16, GSK-3 alpha, GSK-3 beta and PNCK, PRKCG, SRPK1, wherein the GSK-3 beta has a regulation function on a plurality of phosphorylation sites of SYN1 and has statistical significance. The trend of the upstream kinase of SYN1 can be clearly shown through thermal image visualization (FIG. 11B), which is helpful for deep exploration of the phosphorylation control mechanism of SYN1 after spinal cord injury.
3.10 SYN1 highly correlated protein enrichment assay
For further intensive studies of SYN1, whole protein mass spectral data were subjected to correlation analysis, and a protein with a SYN1 pearson correlation coefficient >0.7 was defined as SYN1 highly correlated protein. GO enrichment analysis by using highly related proteins of SYN1 is mainly related to the pathways of organophosphorus synthesis, synaptic tissue, reaction to oxidative stress, axonogenesis, neurotransmitter transmission, neurofilament cytoskeletal tissue and the like in biological processes; enrichment in cell localization is associated with synapses, myelins, axons, microtubules, dendrites, neuronal synapses, etc.; the molecular functions are mainly related to actin, phospholipid binding proteins, etc. (fig. 12A). Enrichment analysis results show that the protein highly related to SYN1 is obviously related to the functions of a nervous system. Spinal cord injury can affect the occurrence and maintenance of axons, thereby affecting neurotransmitter transmission and the formation of synaptic tissue. Spinal cord injury can also lead to an increase in oxidative stress, which can further affect neuronal survival and function.
In addition, enrichment analysis was performed in conjunction with the KEGG database, and the results suggested to be associated with ether lipid metabolism, glycerophospholipid metabolism, and gabaergic synapses (fig. 12B). These results reveal the metabolic pathways and biological functions involved in SYN1 high protein.
3.11 Upregulation of multiple phosphorylation sites of SYN1 following spinal cord injury
SYN1 plays an important functional role in spinal cord injury based on differential analysis of phosphorylation rates and pathway identification of biological processes. In the whole proteome, SYN1 was down-regulated after spinal cord injury (fig. 13A). However, we found that multiple sites of phosphorylation exhibited up-regulation changes, ser62, ser67, ser437, ser510, ser551, ser553, ser568, ser605 (fig. 13B). Therefore, the deep investigation of the phosphorylation control of SYN1 helps to understand the pathophysiological mechanisms of spinal cord injury.
3.12 Western Blot verification of mass spectrometry data accuracy
In identifying SYN1 phosphorylation sites, SYN1 Ser62 is a currently common research site and is one of the key phosphorylation sites of neurologic research. SYN1 and SYN1 Ser62 were therefore selected for Western Blot verification using spinal cord tissue. SYN1 was shown to be down-regulating after the spinal cord injury group at the spinal cord tissue holoprotein level (fig. 14A). Based on the results of the whole proteome, the present example uses the expression level of SYN1 as a calibration level, and uses the expression quantification of SYN1 to compare the phosphorylation level change of the protein site. The results showed that SYN1 Ser62 showed a tendency to be up-regulated in the spinal cord injury group in the case where the expression amounts of SYN1 were uniform (fig. 14B). The results of Western Blot were consistent with the mass spectral data results.
In summary, the invention obtains the data of the proteomics related to the spinal cord injury through the mass spectrometry technology, and identifies the biological signal path related to the spinal cord injury. In addition, through phosphoprotein modification group bioinformatics analysis, the protein related to the synaptic tissue after spinal cord injury is revealed to be significantly changed, SYN1 and SYN1 Ser62 are found to be closely related to spinal cord injury, and meanwhile, a kinase-substrate regulation network of the synaptic tissue after spinal cord injury and SYN1 is constructed. Western Blot confirmed that SYN1 expression was down-regulated in the spinal cord injury group and SYN1 Ser62 expression was up-regulated in the spinal cord injury group, consistent with the results of mass spectrometry analysis. The invention provides the application of the biomarker in the aspects of spinal cord injury diagnosis and prognosis, provides a new target for researching the medicines for treating spinal cord injury, and has important clinical, scientific research and medicine conversion values.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the above-described embodiments and examples, and various changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (5)
1. A spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology, characterized by: the biomarker is selected from one or more of the following: SYN1, SYN1 Ser62.
2. Use of a spinal cord injury biomarker based on proteomics and phosphorylated protein modification spectroscopy in the preparation of a product for diagnosing spinal cord injury, characterized in that: the biomarker is selected from one or more of the following: SYN1, SYN1 Ser62.
3. The use according to claim 2, wherein: the product is a detection reagent, a kit, a microarray or a biochip.
4. The use according to claim 2, wherein: the biomarkers are useful for detection of samples from spinal cord tissue by analytical means of proteomics and phosphorylated protein modification group.
5. Use of a spinal cord injury biomarker based on proteomics and phosphorylated protein modification genetics in the manufacture of a medicament for treating spinal cord injury, characterized in that: the drug targets the biomarker selected from one or more of the following: SYN1, SYN1 Ser62.
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