CN112063603A

CN112063603A - Acetylation modified CNP protein marker molecule related to central nervous senescence and application thereof

Info

Publication number: CN112063603A
Application number: CN202010482928.3A
Authority: CN
Inventors: 季煜华; 陈子鑫; 季秋虹
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-12-11
Anticipated expiration: 2040-06-01
Also published as: CN112063603B

Abstract

The invention discloses an acetylation modified CNP protein marker molecule related to central nervous senescence and application thereof. The protein marker molecule is CNP protein modified by acetylation of lysine at positions 177 and 401. The upregulation of the acetylation modification of the 177 and 401 lysines on the CNP protein is used as a marker for the detection of senescence in cells or individuals. The invention provides an effective research method and a research direction for exploring the aging process, the occurrence of aging-related diseases and the aging research in the future.

Description

Acetylation modified CNP protein marker molecule related to central nervous senescence and application thereof

Technical Field

The invention belongs to the technical field of molecular diagnosis medicine, and relates to an acetylation modified CNP protein marker molecule related to central nerve aging and application thereof.

Background

Aging is mainly manifested by the decline of the biochemical and physiological functions of individuals with age, and is a major risk factor for diseases such as cancer, obesity, type 2 diabetes and the like. With improvements in social healthcare services and systems, the reduction in mortality rates of children and pregnant and lying-in women, and improvements in lifestyle and living standards, the average life expectancy of humans has currently exceeded 60 years. The prolongation of Life greatly affects the population structure, and it is expected that the population number will increase from 6.05 million to 20 million around the World between 2000 and 2050 years for 60 years and beyond (World Health Organization, "Life exhibition in creatable by 5 years site 2000, but Health in interests past," http:// www.who.int/media/news/reviews/2016/Health in interests-experience/en.). An increase in aging population will have profound effects on social and economic activities. Although we cannot inhibit the aging process, finding the occurrence of aging as early as possible and delaying aging through certain intervention or treatment will help to reduce the risk of aging-related diseases, improve the quality of life of the elderly, and reduce the burden on families and society. For aging research, a problem to be solved is to find markers (Burkle, Grune, Gonos, & Bohr,2015) that can be quantitatively analyzed for aging, which is also necessary for evaluating the effect of anti-aging treatment.

It is generally believed that aging in mammals has the following 9 characteristics: genome instability, loss of telomeres, epigenetic changes, decreased protein stability, dysregulation of nutrient perception, mitochondrial dysfunction, cellular senescence, stem cell failure, and alterations in cell-cell communication (Lopez-Oti i n, Blasco, Partridge, Serrano, & Kroemer, 2013). Markers that have been found to reflect cellular senescence include: phosphorylation of histone h2a.x (Ser139) (also known as γ -h2a.x, a marker of DNA damage), increased expression of p16INK4A, loss of expression of lamin B1, increased lysosomal content and altered lysosomal activity, among others. In addition, senescent cells are also characterized by extensive chromatin remodeling, most notably the formation of senescence-associated heterochromatin clusters (SAHF) (Freund, Laberge, Demaria, & Campisi, 2012; LaPak & Burd, 2014; Nacarelli, Liu, & Zhang, 2017; Rogakou, Boon, Redon, & Bonner, 1999). Tissue levels are the level of senescence most determined by the overall level of metabolites, such as total homocysteine (tHcy), total Cholesterol, very low, High density lipoprotein density lipoproteins, triglycerides, etc. (Arnesen et al, 1995; Cullen, 2000; Expert Panel on protection & Treatment of High Blood Cholesterol in, 2001; Grundy et al, 2004; Manlio et al, 1992; Verhoef et al, 1996). Partial central nervous system marker proteins may also be used as aging markers, such as beta-amyloid 42(A beta 42), total tau, phosphorylated tau, and F-isoprostane (F2-iso) (Blennow, Vanmechelen, & Hampel, 2001; Galasko et al, 1998; Kim, Jung, Paeng, Kim, & Chung, 2004; Poitou et al, 2005), and the like.

The protein is the material basis of life activity, and the physicochemical properties of the primary structure and the folded higher structure of the protein are the basis of the activity and stability of the protein. Protein post-translational modification (PTM) is the binding of specific chemical groups to amino acid side chains in an enzyme-dependent or enzyme-independent manner, thereby affecting protein structure and function. Enzymatic PTMs include phosphorylation, glycosylation, acetylation, methylation, sumoylation, palmitoylation, biotinylation, ubiquitination, nitration, chlorination, and oxidation/reduction. Whereas non-enzymatic PTMs include glycosylation, nitrosylation, oxidation/reduction, acetylation, and succination (Witze, Old, curing, & Ahn, 2007).

Changes in post-translational modifications of proteins during senescence have been noted for a long time, and it was found that the accumulation of abnormal forms of enzymes is associated with the post-translational modifications of proteins with increasing age, and the progressive loss of enzymes that degrade these modifications with increasing age (Stadtman, 1988). Recent studies have also suggested that changes in post-translational modifications of proteins and the accumulation of some covalently modified proteins are markers of biological senescence (Vanhooren et al, 2015). The association of enzyme-dependent and enzyme-independent post-translational glycosylation modification of serum proteins with senescence was first fully explored in the "Mark-Age" project completed by european scientists in 2015 (Burkle et al, 2015). In addition to glycosylation, ubiquitination, methylation and acetylation modifications of proteins may also be associated with senescence (Santos & Lindner, 2017). Accurate analysis of the PTM modification site will therefore provide a possible marker for early diagnosis, prognosis, and detection of the effect of treatment.

Disclosure of Invention

The invention aims to provide an acetylation modified CNP protein marker molecule related to central nervous senescence.

The invention also aims to provide application of the acetylation modified CNP protein marker molecule related to central nervous senescence.

In order to achieve the purpose, the invention is realized by the following technical scheme:

an acetylation modified CNP protein marker molecule related to central nervous senescence is a CNP protein with the 177 th lysine and the 401 th lysine being modified by acetylation.

The amino acid sequence of the CNP protein is as follows:

MNTSFTRKSHTFLPKLFFRKMSSSGAKEKPELQFPFLQDEDTVATLHECKTLFILRGLPGSGKSTLARLILEKYHDGTKMVSADAYKIIPGSRADFSEAYKRLDEDLAGYCRRDIRVLVL

EKDFLPLYFGWFLTKKSSETLRKAGQVFLEELGNHKAFKKELRHFISGDEPKEKLELVSYFGKRPPGVLHCTTKFCDYGKAAGAEEYAQQEVVKRSYGKAFKLSISALFVTPKTAGAQVVLTDQELQLWPSDLDKPSASEGLPPGSRAHVTLGCAADVQPVQTGLDLLDILQQVKGGSQG

the acetylation modified CNP protein marker molecule related to central nervous senescence is applied to cell, tissue, organ or individual senescence degree detection; preferably comprising the steps of:

(1) extracting, purifying and quantifying animal cell or tissue protein;

(2) carrying out enzymolysis on the purified protein;

(3) enriching acetylation modified peptide fragments by using a specific antibody;

(4) quantitatively analyzing acetylation modification of cells or tissue proteins based on a liquid chromatography-mass spectrometry technology;

(5) the degree of aging of a cell, tissue, organ or individual is evaluated by quantitative analysis results.

The animal in the step (1) is a mammal.

The mammals include mice, human beings, monkeys, chimpanzees and the like.

The mice include mice and rats.

The mouse is preferably a C57BL/6 mouse.

The monkey is preferably a cynomolgus monkey.

The orangutan is preferably Sumendortan.

The tissue in step (1) is preferably brain tissue.

The method for quantification described in step (1) is preferably a Bradford method.

The enzymolysis in the step (2) is preferably realized by a FASP enzymolysis method.

The specific antibody in step (3) is preferably an anti-acetyl lysine antibody.

The enrichment described in step (3) is preferably achieved by immunoaffinity precipitation.

The quantitative analysis process described in step (4) preferably comprises the steps of:

1) resuspending the enriched acetylated modified peptide fragment, and separating;

2) then, carrying out mass spectrum identification to obtain mass spectrum original data;

3) and (4) performing library searching on the mass spectrum original data through Maxquant software so as to obtain a peptide fragment information list.

The separation described in step 1) is preferably carried out by an easy nano LC1000 system.

The separation conditions are preferably as follows: the fractions were carried out at 500 nL/min by using solution A and solution B as mobile phases and a C18 column (75 μm. times.15 cm, 3 μm) with a separation gradient: 1% B to 3% B in 2 minutes, 3% B to 8% B in the next 8 minutes, 8% B to 20% B in the next 45 minutes, 20% B to 30% B in the next 12 minutes, 30% B to 90% B in the next 1 minute, and then stabilize for 90% B7 minutes; the liquid A is 0.1% (v/v) formic acid-acetonitrile (the solvent is acetonitrile, the solute is formic acid), the liquid B is 0.1% (v/v) formic acid-water (the solvent is water, the solute is formic acid), and the total volume percentage of the liquid A and the liquid B is 100%.

The mass spectrometric identification described in step 2) is preferably carried out using an Orbitrap Fusion Lumos mass spectrometer.

The mass spectrometric identification conditions described in step 2) are preferably mass spectrometric identification in the form of DDA.

The parameters for searching the library in the step 3) are as follows: the sample type is standard, and the multiplex is 1; the protein digestive enzymes are: trypsin; fixing and modifying: carbammidomethyl (C); the variable modifications are acetyl (K), Deamidino (N), Deamidino (NQ); the minimum peptide length is 6; the mismatch rate (FDR) threshold of the peptide fragment and the protein is less than 0.01; the variable modifications used for Protein quantification are oxidation (M), Acetyl (Protein N-term), peptide fragment unique.

The criteria for the evaluation described in step (5) are preferably as follows: the total acetylation modification upregulation degree and the senescence degree of the acetylation modified CNP protein marker molecules related to central nervous senescence are positively correlated, namely, the acetylation modification upregulation degree and the senescence degree of the 177 th lysine and the 401 th lysine are positively correlated.

The acetylation modified CNP protein marker molecule related to central nerve aging is applied to the preparation of a kit for predicting aging-related degenerative diseases.

In the application, the aging degree of the biological sample is judged by determining the molecular acetylation modification degree of the marker molecules in the biological sample.

The determination of the degree of molecular acetylation modification of the CNP protein marker molecule can be achieved by a method or a device for determining the expression level of the related molecule and the acetylation level of the protein in a sample.

The acetylation modified CNP protein marker molecule related to central nerve aging is applied to the preparation of medicines for preventing and/or treating degenerative diseases related to aging.

The application is to use the acetylation modified CNP protein marker molecule related to central nervous senescence as a drug target.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a biomarker related to aging and a detection method, and CNP (2', 3' -cyclic nucleotide 3' -phosphodiesterase) is used for acetylation modification up-regulation of K177 and K401 sites in aged mouse brain and is used as a marker for cell or individual aging detection. The invention provides an effective research method and a research direction for exploring the aging process, the occurrence of aging-related diseases and the aging research in the future.

Drawings

Fig. 1 is a technical route diagram of the present invention.

FIG. 2 is a secondary spectrum of the peptide fragments LK (ac) PGLEK and GK (ac) PVPIHGSR in which CNP K177 and K401 are located, which is identified by mass spectrometry; wherein a is a secondary spectrum of the peptide fragment of K177 in the sample Y3; b is a secondary spectrum detected by K177 in sample O3; c is a secondary spectrum detected by K401 in a Y3 sample.

FIG. 3 is a graph of the relative quantification of the acetylation of CNP K177 and K401 in different groups of mouse brains in mass spectrometry; wherein a is a relative quantitative result; b is a histogram of the corresponding statistical results.

FIG. 4 is an ion flow diagram and a peak area statistical result diagram of a peptide fragment LK (ac) PGLEK in which CNP K177 is located in each sample identified by mass spectrometry; wherein a is an ion flow graph of the peptide fragment LK (ac) PGLEK in each sample; b is the corresponding peak area of the peptide fragment LK (ac) PGLEK in each sample; c is a histogram of statistical results of the b picture.

FIG. 5 is an ion flow diagram and a peak area statistical result diagram of a peptide segment GK (ac) PVPIHGSR in which CNP K401 is identified by mass spectrometry in each sample; wherein a is an ion flow diagram of a peptide segment GK (ac) PVPIHGSR in each sample; b is the corresponding peak area of peptide segment GK (ac) PVPIHGSR in each sample; c is a histogram of statistical results of the b picture.

Fig. 6 is an ion flow graph and a peak area statistical result graph of the CNP nonacetylated peptide segment GGSQGEAVGELPR identified by mass spectrometry in each sample; wherein a is the ion flow map of peptide segment GGSQGEAVGELPR in each sample; b is the corresponding peak area of peptide fragment GGSQGEAVGELPR in each sample; c is a histogram of statistical results of the b picture.

FIG. 7 is a graph showing the results of CoIP verification of mice in different groups; wherein a is a co-immunoprecipitation and Western blot verification result graph; b is a histogram of statistical results of the a diagram.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. The technical route of the invention is shown in figure 1.

The reagents and the preparation method related in the embodiment of the invention are as follows:

1mol/L Tris HCl pH 8.0 solution: 12.11g Tris base (Tris-base) was dissolved in pure water, adjusted to pH 8.0 with concentrated hydrochloric acid, and made up to 100ml ultrapure water.

4% chloral hydrate: 0.4g of chloral hydrate was weighed out, dissolved in distilled water and diluted to 10 ml with distilled water.

8mol/L urea buffer: 48.048g of urea granules were weighed out, 5ml of Tris HCl (pH 8.0) solution with a concentration of 1mol/L was added, and the volume of ultrapure water was adjusted to 100 ml.

1mol/L Dithiothreitol (DTT) solution: weighing 15.42g of dithiothreitol, adding ultrapure water to dilute the dithiothreitol into 100ml, dividing the dithiothreitol into small parts, and storing the small parts at-20 ℃.

1mol/L Iodoacetamide (IAA) solution: 0.185g IAA was weighed into 1ml urea buffer with a concentration of 8mol/L until complete dissolution.

50mmol/L NH₄HCO₃: 2g of ammonium bicarbonate is weighed and ultrapure water is added to the solution until the volume is 50 ml.

1 × PBS: weighing 8g NaCl, 0.2g KCl and 1.44g Na₂HPO₄And 0.24g KH₂PO₄Dissolving in 800ml distilled water, adjusting pH value of the solution to 7.4 with HCl, and adding distilled water to constant volume to 1L.

0.05% PBST: 0.5mL of Tween-20 was aspirated, and 1L of PBS buffer was added to the solution to obtain a volume of 1L.

1% acetonitrile, 0.1% trifluoroacetic acid solution (v/v): acetonitrile and trifluoroacetic acid are added into ultrapure water according to the volume ratio and then are shaken up, and the final volume is 500 ml.

40% acetonitrile, 0.1% trifluoroacetic acid solution (v/v): acetonitrile and trifluoroacetic acid are added into ultrapure water according to the volume ratio and then are shaken up, and the final volume is 500 ml.

NETN buffer solution: 50mM Tris, 1mM EDTA, 100mM NaCl, 0.5% Nonidet P-40 (ethylphenylpolyethylene glycol), pH 8.0.

ETN buffer 100mM NaCl, 1mM EDTA, 50mM Tris, pH 8.0.

Example 1 sample preparation prior to Mass Spectrometry identification

Extraction of C57BL/6 mouse brain protein

1) The C57BL/6 mice (Experimental animals center of Guangzhou university of traditional Chinese medicine) are divided into three groups, including three young mice (Y1 group, Y2 group, Y3 group, mouse age 2 months) and three old mice (O1 group, O2 group, O3 group, mouse age 16 months); each group had 6 mice.

2) Each mouse is anesthetized by 4% chloral hydrate intraperitoneal injection according to 0.1mL/kg, the abdominal cavity is dissected, after the heart is exposed, the heart is perfused by 1 XPBS buffer solution, and the brain tissue is dissected and weighed;

3) placing the brain tissue in 8mol/L urea buffer solution (the final concentration of the brain tissue is 20mg/mL), and homogenizing by a glass homogenizer to obtain brain tissue homogenate;

4) homogenates of brain tissue are sonicated: the power is 30w, the crushing lasts for 10s, the interval is 10s, and 5 cycles are carried out to obtain brain tissue lysate;

5) centrifuging the brain tissue lysate at 13000g for 30min for 2 times; taking the supernatants of the two times, and mixing;

6) protein concentration was quantified using the Bradford method (Bradford protein concentration assay kit purchased from petunia, product No. P0006; the specific operation steps are carried out according to the instruction).

Enzymatic hydrolysis of FASP

1) Respectively taking the amount of the cerebroprotein of each group of young and old mice to be 2mg, and adding 8mol/L urea buffer solution to 2mL (namely adjusting the concentration of the cerebroprotein to be 1.0mg/mL) to obtain protein suspension;

2) the protein suspension was added to a 10kD 4mL ultrafiltration tube (Millipore Ultrafiltration centrifuge tube 4mL/10 kD)

Ultra-4, 2mg brain protein amount/tube), sleeving an ultrafiltration tube in a 15ml centrifuge tube, centrifuging at 4000g and 24 ℃ until the liquid in the upper chamber of the tube (the ultrafiltration tube is the upper chamber) is less than 100 muL；

3) Adding 1mol/L DTT solution into the upper chamber until the final concentration of DTT is about 20mmol/L, shaking, mixing, air-bathing at 37 deg.C for 1.5h, centrifuging at 4000g and 24 deg.C until the volume of liquid in the upper chamber is less than 100 μ L;

4) pouring out the liquid in the lower chamber of the ultrafiltration tube, adding 1mol/L IAA solution into the upper chamber until the final concentration of the IAA is about 0.1mol/L, shaking, mixing uniformly, keeping out of the sun for 20min at normal temperature, centrifuging under the same conditions until the residual liquid is less than 50 mu L;

5) 50mmol/L NH for upper chamber protein₄HCO₃Washing and replacing the solution with 50mmol/L NH₄HCO₃After that, pancreatin was added in a ratio of 1:100, i.e. 50mmol/L NH per tube at 2mL (same volume of the original protein)₄HCO₃Adding 20 mu g of pancreatin; the lower chamber was also charged with 1ml of 50mmol/L NH₄HCO₃(ii) a Incubating for 16h at 37 ℃, replacing a new collecting pipe to collect centrifuged liquid, namely a total peptide fragment mixture, and pumping the peptide fragments by a freeze dryer;

6) desalting the peptide segment: the resulting dried peptide fragment was dissolved in phase A (1% acetonitrile, 0.1% trifluoroacetic acid in water) and loaded onto an equilibrated 4.6mmC18 analytical column (Agilent 1200 HPLC). Washing 1ml/min of phase A for 10 min to remove salts and other impurities from the sample, eluting with 1ml/min of phase B (40% acetonitrile, 0.1% trifluoroacetic acid solution) for 5 min and collecting the elution peak, and freeze-drying the collected eluate to obtain purified peptide fragment for next immunoaffinity precipitation.

3. Immunoaffinity precipitation enrichment of acetylated peptide fragments

1) Pipetting 30. mu.L of antibody-coated agarose beads (Pan acetylated antibody-conjugated resin, Pan anti-acetyllysine conjugated agarose beads, PTM BIO, PTM-104) with a pipette, placing in a small filter tube and sleeving on an EP tube, washing the agarose beads with pre-cooled 1 XPBS buffer, centrifuging 500g for 15s, and repeating three times;

2) dissolving the dried peptide fragment with NETN buffer solution, adding into the washed agarose beads, plugging the lower surface of the filter tube with a plug, sealing the cover with a sealing film, fixing on a rotary mixer, and rotationally mixing at 4 deg.C overnight;

3) taking the incubated agarose beads out of the refrigerator the next day, taking a clean EP tube, removing a plug below the filter tube, and centrifuging the liquid into the EP tube; adding 0.5ml of NETN buffer solution into a filter tube, reversing the upside down and mixing the mixture evenly, 500g of the mixture, centrifuging the mixture for 15s, and washing the mixture twice; adding 0.5ml of ETN buffer solution into a filter tube, reversing the upper part and the lower part, uniformly mixing, washing twice under the same centrifugal condition; adding 0.5ml of deionized water or ultrapure water into the filter tube, and washing twice according to the steps; adding 100 μ L of Elution Buffer (Elution Buffer) into the filter tube, inverting for 1min, 500g, centrifuging for 15s, repeating twice again, and collecting the volume of 300 μ L in total;

4) the collected liquid is drained and stored at-20 ℃ to obtain the acetylated peptide fragment.

Example 2 LC-MS/MS identification and data searching

LC-MS/MS identification

1) The enriched peptides were not fractionated and were directly subjected to nanoLC-MS/MS analysis using 0.1% formic acid-acetonitrile (a) and 0.1% formic acid-water (B) as mobile phases, and the peptides of each sample were resuspended in mass spectrometry mobile phase a solution and separated by EasyNano LC1000 system (San Jose, Thermo Fisher) at 500 nL/min on a C18 column (75 μm × 15cm, 3 μm); the flow-through linear gradient is shown in table 1:

TABLE 1 LC-MS/MS elution conditions

Time/min	Flow rate (nL/min)	A％(v/v)	B％(v/v)
				0:00	500	99	1
2:00	500	97	3
				10:00	500	92	8
55:00	500	80	20
				67:00	500	70	30
68:00	500	10	90
				75:00	500	10	90

2) Mass spectrometric identification of peptide fragments was performed using an Orbitrap Fusion Lumos (Bremen, Thermo Fisher) mass spectrometer by means of DDA: the scanning period is 3s, the MS1 parent ion scanning range is m/z 350-: starting at 100 m/z. Other parameter settings were as follows: the MS1 and MS2 resolutions were set to 120K and 30K, respectively; with Automatic Gain Control (AGC), MS1 and MS2 are 1e6 and 1e5, respectively; the ion screening window is a quadrupole rod, and is 1.6 m/z; fragmentation mode: HCD, Normalized Collision Energy (NCE) 32; the dynamic exclusion time was 20 s. Secondary maps of the peptide fragments LK (ac) PGLEK and GK (ac) PVPIHGSR in which the 177 th and 401 th lysines of CNP are identified by mass spectrum are shown in FIG. 2. The peptide segment is broken into ions with different mass-to-charge ratios after being detected by mass spectrometry of different b and y ion pairs, and the theoretical mass of the amino acid composition of the peptide segment is met.

2. Mass spectrometry result library search

Mass spectra raw data each set of treatments was pooled by Maxquant (version 1.6.3.3) software, protein sequence database from Uniprot Mus musculus (mouse) containing 10090 protein sequences, the pooling parameters were set to: 1) the sample type is standard, and the multiplex is 1; 2) the protein digestive enzymes are: trypsin; 3) fixing and modifying: carbammidomethyl (C); 4) the variable modifications are acetyl (K), Deamidino (N), Deamidino (NQ); 5) the minimum peptide length is 6; 6) the mismatch rate (FDR) threshold of the peptide fragment and the protein is less than 0.01; 7) the parameters of modified peptide fragment for Protein quantification are oxidation (M), Acetyl (Protein N-term), and peptide fragment for quantification is unique, and the result is used as the change of Protein expression.

Example 3 Mass Spectrometry results with increased CNP acetylation levels

In the Maxquant library results table, the intensity value of each site in the Acetyl (K) sites. txt table is corrected by the total ion current intensity between each sample, and the corrected intensity value is used as the quantification of the acetylated site in each sample. Similarly, each protein intensity value in the protein groups. txt table was corrected by the total ion flux intensity between each sample, and the corrected intensity value was used as a quantitative value for the CNP protein expression level in each sample. The multiple of the intensity difference of the CNP acetylation sites and the multiple of the protein expression difference are counted by using GraphPad Prism software, and the corresponding p-value is tested by t-tests. Meanwhile, the change of acetylation modification was corrected by the change of abundance of non-acetylated proteins to eliminate the change of acetylation modification caused by the change of protein expression amount, and the result is shown in fig. 3. The O-to-Y ratios of peptide fragments of acetylated K177 and K401 are respectively 2.19 and 3.19(p is less than 0.05), and the O-to-Y ratios of acetylated K177 and K401 are respectively 2.57 and 3.7(p is less than 0.05 and p is less than 0.01) after protein abundance correction.

In order to further analyze the changes of expression of the CNP proteins acetylated K177 and K401 in O and Y, an xcalibur software (Thermo scientific) is adopted to directly find out an extracted ion flow graph of the CNP K177 and K401 acetylated peptide segments in the original data. Fig. 4 and 5 show the peak area plots, peak area statistics, and histograms for CNP K177 and K401 acetylated peptide fragments in each sample, respectively, and the results show that acetylation of K177 is increased in O relative to Y (p < 0.0001). Fig. 6 is a peak plot of the nonacetylated peptide fragment GGSQGEAVGELPR in CNP in each sample, which was significantly down-regulated in O (p <0.05) relative to the Y sample.

Example 4 verification of Mass Spectrometry results by Co-immunoprecipitation (CoIP)

160. mu.g of brain protein samples from aged (O) and young (Y) mice were prepared, 20. mu.L of Antibody-coated Agarose Beads (Pan PTM Antibody Conjugated Agarose Beads, Lysine acetic, PTM BIO, PTM-104) were pipetted separately, washed 3 times with PBS, and added to the sample proteins followed by rotary incubation at 4 ℃ overnight. The next day, the cells were removed from the freezer, centrifuged at 800g for 5 minutes, the supernatant was discarded, the pellet was washed 5 times with 0.05% PBST, the target protein was eluted with 0.2% formic acid, the supernatant was centrifuged, and the supernatant was vacuum-dried and then subjected to Western blot analysis using a 2 Xloading buffer in a boiling water bath for 5 minutes, using a primary antibody against CNP (Cell Signaling Technology,5664S) and a secondary antibody against mouse IgG (CST, 7076S). Results as shown in fig. 7, CNP bars enriched with pan-acetylated antibodies from groups O and Y were corrected by CNP from Input, respectively, and the results showed a significant upregulation of CNP acetylation.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> acetylation modified CNP protein marker molecule related to central nervous senescence and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 420

<212> PRT

<213> mouse (Mus musculus)

<220>

<223> amino acid sequence of CNP protein

<400> 1

Met Asn Thr Ser Phe Thr Arg Lys Ser His Thr Phe Leu Pro Lys Leu

1 5 10 15

Phe Phe Arg Lys Met Ser Ser Ser Gly Ala Lys Glu Lys Pro Glu Leu

20 25 30

Gln Phe Pro Phe Leu Gln Asp Glu Asp Thr Val Ala Thr Leu His Glu

35 40 45

Cys Lys Thr Leu Phe Ile Leu Arg Gly Leu Pro Gly Ser Gly Lys Ser

50 55 60

Thr Leu Ala Arg Leu Ile Leu Glu Lys Tyr His Asp Gly Thr Lys Met

65 70 75 80

Val Ser Ala Asp Ala Tyr Lys Ile Ile Pro Gly Ser Arg Ala Asp Phe

85 90 95

Ser Glu Ala Tyr Lys Arg Leu Asp Glu Asp Leu Ala Gly Tyr Cys Arg

100 105 110

Arg Asp Ile Arg Val Leu Val Leu Asp Asp Thr Asn His Glu Arg Glu

115 120 125

Arg Leu Asp Gln Leu Phe Glu Met Ala Asp Gln Tyr Gln Tyr Gln Val

130 135 140

Val Leu Val Glu Pro Lys Thr Ala Trp Arg Leu Asp Cys Ala Gln Leu

145 150 155 160

Lys Glu Lys Asn Gln Trp Gln Leu Ser Ala Asp Asp Leu Lys Lys Leu

165 170 175

Lys Pro Gly Leu Glu Lys Asp Phe Leu Pro Leu Tyr Phe Gly Trp Phe

180 185 190

Leu Thr Lys Lys Ser Ser Glu Thr Leu Arg Lys Ala Gly Gln Val Phe

195 200 205

Leu Glu Glu Leu Gly Asn His Lys Ala Phe Lys Lys Glu Leu Arg His

210 215 220

Phe Ile Ser Gly Asp Glu Pro Lys Glu Lys Leu Glu Leu Val Ser Tyr

225 230 235 240

Phe Gly Lys Arg Pro Pro Gly Val Leu His Cys Thr Thr Lys Phe Cys

245 250 255

Asp Tyr Gly Lys Ala Ala Gly Ala Glu Glu Tyr Ala Gln Gln Glu Val

260 265 270

Val Lys Arg Ser Tyr Gly Lys Ala Phe Lys Leu Ser Ile Ser Ala Leu

275 280 285

Phe Val Thr Pro Lys Thr Ala Gly Ala Gln Val Val Leu Thr Asp Gln

290 295 300

Glu Leu Gln Leu Trp Pro Ser Asp Leu Asp Lys Pro Ser Ala Ser Glu

305 310 315 320

Gly Leu Pro Pro Gly Ser Arg Ala His Val Thr Leu Gly Cys Ala Ala

325 330 335

Asp Val Gln Pro Val Gln Thr Gly Leu Asp Leu Leu Asp Ile Leu Gln

340 345 350

Gln Val Lys Gly Gly Ser Gln Gly Glu Ala Val Gly Glu Leu Pro Arg

355 360 365

Gly Lys Leu Tyr Ser Leu Gly Lys Gly Arg Trp Met Leu Ser Leu Thr

370 375 380

Lys Lys Met Glu Val Lys Ala Ile Phe Thr Gly Tyr Tyr Gly Lys Gly

385 390 395 400

Lys Pro Val Pro Ile His Gly Ser Arg Lys Gly Gly Ala Met Gln Ile

405 410 415

Cys Thr Ile Ile

420

Claims

1. An acetylation modified CNP protein marker molecule related to central nervous senescence, which is characterized in that: the acetylation modified CNP protein marker molecules related to the central nervous senescence are CNP proteins of which the 177 th lysine and the 401 th lysine are modified by acetylation.

2. The cns senescence-associated acetylated modified CNP protein marker molecule of claim 1, wherein: the amino acid sequence of the CNP protein is shown as SEQ ID NO. 1.

3. Use of the acetylation modified CNP protein marker molecule related to central nervous senescence of claim 1 or 2 in the detection of the degree of senescence in cells, tissues, organs or individuals.

4. Use according to claim 3, characterized in that it comprises the following steps:

(1) extracting and purifying proteins of animal cells or tissues;

(2) carrying out enzymolysis on the purified protein;

5. Use according to claim 4, characterized in that:

the animals in the step (1) include mice, humans, monkeys and chimpanzees;

the tissue in the step (1) is brain tissue;

the enzymolysis in the step (2) is realized by adopting an FASP enzymolysis method;

the enrichment described in step (3) is achieved by immunoaffinity precipitation.

6. Use according to claim 5, characterized in that:

the mice comprise mice and rats;

the monkey is a cynomolgus monkey;

the orangutan is a Sumendortan.

7. Use according to claim 4, characterized in that:

the specific operation of the quantitative analysis process in the step (4) is as follows:

3) searching the mass spectrum original data through Maxquant software to obtain a peptide fragment information list;

the criteria for the evaluation described in step (5) are as follows: the acetylation modification up-regulation degree of lysine 177 and lysine 401 of CNP protein is positively correlated with senescence degree.

8. Use according to claim 7, characterized in that:

the separation in step 1) is carried out by an easy Nano LC1000 system;

the mass spectrometric identification described in step 2) uses an Orbitrap Fusion Lumos mass spectrometer;

the parameters for searching the library in the step 3) are as follows: the sample type is standard, and the multiplex is 1; the protein digestive enzymes are: trypsin; fixing and modifying: carbammidomethyl C; the variable modification is Acetyl K, Deamidination N, Deamidination NQ; the minimum peptide length is 6; the mismatching rate threshold of the peptide fragment and the protein is less than 0.01; peptide fragments used for Protein quantification were variably modified to Oxidation M, Acetyl Protein N-term, and peptide fragments to unique.

9. Use of the acetylated modified CNP protein marker molecule of claim 1 or 2 in the preparation of a kit for predicting senescence-associated degenerative diseases.

10. Use of the acetylated modified CNP protein marker molecule of claim 1 or 2 as a drug target in the prevention and/or treatment of degenerative diseases associated with aging.