CN112063599B

CN112063599B - Acetylation modified SIRT2 protein marker molecule related to central nervous senescence and application thereof

Info

Publication number: CN112063599B
Application number: CN202010484549.8A
Authority: CN
Inventors: 季秋虹; 季菊玲; 陈子鑫; 季煜华
Original assignee: Affiliated Hospital of Nantong University
Current assignee: Affiliated Hospital of Nantong University
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2023-01-17
Anticipated expiration: 2040-06-01
Also published as: CN112063599A

Abstract

The invention discloses an acetylation modified SIRT2 protein marker molecule related to central nervous senescence and application thereof. The protein marker molecule is SIRT2 protein with acetylation modified lysine at the 126 th site and the 55 th site. The upregulation of lysine acetylation modification at 55 and 126 positions on SIRT2 protein 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.

Description

Acetylation modified SIRT2 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 particularly relates to an acetylation modified SIRT2 protein marker molecule related to central nervous senescence 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 and type 2 diabetes. With improvements in social health care services and systems, and with the reduction in mortality rates of children and pregnant and parturient 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 structure of the population, and it is expected that the population number will increase from 6.05 million to 20 million around the World from 2000 to 2050 (World Health Organization, "Life expectance in cultured by 5years approach 2000, but Health in evaluation experience," http:// www. Who. Int/media/news/reuses/2016/Health in evaluation-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 (Ser 139) (also known as γ -h2a.x, DNA damage marker), increased expression of p16INK4A, loss of lamin B1 expression, increased lysosomal content, altered lysosomal activity, and the like. In addition, aging cells are also characterized by extensive chromatin remodeling, most notably the formation of aging-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 as 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. Some central nervous system marker proteins may also be used as aging markers, such as β -amyloid 42 (a β 42), total tau, phosphorylated tau, and F-isoprostane (F2-iso) (Blennow, vanmechlen, & Hampel,2001, galasko et al, 1998, kim, jung, paeng, kim, & Chung,2004, poitouu et al, 2005).

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, resing, & 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 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 SIRT2 protein marker molecule related to central nervous senescence.

The invention also aims to provide application of the acetylation modified SIRT2 protein marker molecule related to central nervous senescence.

The purpose of the invention is realized by the following technical scheme:

an acetylation modified SIRT2 protein marker molecule related to central nervous senescence is SIRT2 protein with 126 th lysine and 55 th lysine being modified by acetylation.

The SIRT2 protein is preferably an amino acid sequence of a SIRT2 protein from a rat, and the amino acid sequence is preferably as follows:

the acetylation modified SIRT2 protein marker molecule related to the central nervous senescence is applied to the detection of the senescence degree of cells, tissues, organs or individuals; preferably comprising the steps of:

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

(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 the cell, tissue, organ or individual is evaluated by quantitative analysis of the results.

The animal in the step (1) is a mammal, including a mouse, a human, a monkey, an orangutan 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 enzymolysis in the step (2) is preferably realized by an FASP enzymolysis method.

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

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

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) searching the library for the mass spectrum raw 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 in step 1) is specifically carried out by taking the liquid A and the liquid B as mobile phases and passing the mobile phases through a C18 column (3 μm,75 μm × 15 cm) at 500nL per minute, and the separation gradient is as follows: 1-to 3-percent B in 2 minutes, 3-to 8-percent B in the next 8 minutes, 8-to 20-percent B in the next 45 minutes, 20-to 30-percent B in the next 12 minutes, 30-to 90-percent B in the next 1 minute, and then stabilize 90-percent B for 7 minutes; the A liquid is 0.1% (v/v) formic acid-acetonitrile (the solvent is acetonitrile, the solute is formic acid), the B liquid is 0.1% (v/v) formic acid-water (the solvent is water, the solute is formic acid), and the total volume percentage of the A liquid and the B liquid is 100%.

The mass spectrometric identification described in step 2) preferably uses an Orbitrap Fusion Lumos mass spectrometer.

The mass spectrometric identification described in step 2) is preferably carried out in the manner 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 modification is Acetyl (K), deamidation (N), deamidation (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 peptide fragment used for the quantification of acetylated proteins was modified to Acetyl (K), and the peptide fragment was unique.

The criteria for the evaluation described in step (5) are as follows: acetylation modification up-regulation degree of lysine 126 and lysine 55 of SIRT2 protein and senescence degree are positively correlated.

The acetylation modified SIRT2 protein marker molecule related to central nervous senescence is applied to preparation of a kit for predicting senescence-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 modification by molecular acetylation of the marker molecule may be achieved by a method or apparatus for determining the expression level of the relevant molecule and the acetylation level of the protein in the sample.

The acetylation modified SIRT2 protein marker molecule related to the central nervous senescence is applied to the preparation of medicines for preventing and treating degenerative diseases related to senescence.

The application is to apply the acetylation modified SIRT2 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 biological marker related to aging and a detection method, wherein SIRT2 (nicotinamide adenine dinucleotide dependent protein deacetylase) is used for acetylation modification up-regulation of K55 and K126 sites in aged mouse brain, and can be 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 scheme of the present invention.

FIG. 2 is a secondary spectrum of peptide segments NLFTQTLGLGSQK (ac) ER and K (ac) HPEPFFALAK in which SIRT 2K 55 and K126 are identified by mass spectrometry; wherein a is a secondary spectrogram of a peptide fragment in which K55 is located; b is a secondary spectrum detected by K126 in an O1 sample; c is a secondary spectrum detected by K126 in the Y1 sample.

FIG. 3 is a graph of the relative quantitative results of the acetylation of SIRT 2K 126 and K55 in different groups of mouse brains in mass spectrometric identification results; wherein a is a relative quantitative result graph; 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 segment K (ac) HPEPFFALAK in which SIRT 2K 126 is identified by mass spectrometry in each sample; wherein a is an ion flow diagram of a peptide segment K (ac) HPEPFFALAK in each sample; b is the corresponding peak area of peptide fragment K (ac) hpepffalk in each sample; c is a histogram of statistical results of the b picture.

FIG. 5 is an ion flow graph and a peak area statistical result graph of the SIRT2 non-acetylated peptide fragment ELYPGQFRKPTICHYFIR identified by mass spectrum in each sample; wherein) is an ion flow graph of the peptide segment ELYPGQFRKPTICHYFIR in each sample; b is the corresponding peak area of the peptide fragment ELYPGQFKPTICHYFIR in each sample; c is a histogram of statistical results of the b picture.

FIG. 6 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; b is a histogram of statistical results of the a diagram.

FIG. 7 is a diagram showing the result of sequence conservation analysis of SIRT2 proteins K55 and K126 in different species.

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.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The reagent and the preparation method thereof related to 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 are weighed, 5ml of Tris HCl (pH 8.0) solution with the concentration of 1mol/L is added, and the volume of ultrapure water is 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 NH ₄ HCO ₃ : 2g of ammonium bicarbonate is weighed, and ultrapure water is added to the solution until the volume is 50ml.

1 × PBS: weighing 8g NaCl, 0.2g KCl and 1.44g Na ₂ HPO ₄ And 0.24g KH ₂ PO ₄ Dissolving in 800ml distilled water, adjusting pH value of the solution to 7.4 with HCl, and adding distilled water to constant volume of 1L.

0.05% PBST: absorbing 0.5mL of Tween-20, and adding 1 XPBS buffer solution to make the volume constant to 1L.

1% acetonitrile, 0.1% trifluoroacetic acid solution (v/v): acetonitrile and trifluoroacetic acid were added to ultrapure water in a volume ratio, followed by shaking to give a final volume of 500ml.

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 500ml.

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

C57BL/6 mouse brain protein extraction

1) Grouping C57BL/6 mice (Experimental animals center of Guangzhou university of traditional Chinese medicine) into three groups, including three young mouse groups (Y1 group, Y2 group, Y3 group, mouse age 2 months) and three old mouse groups (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 according to 20mg/ml, and homogenizing by a glass homogenizer;

4) Homogenates of brain tissue are sonicated: carrying out ultrasonic crushing for 10s at the power of 30w, at the interval of 10s and 5 cycles to obtain a 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) Bradford quantification (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 calculating the brain protein amount of 2mg of young mice and old mice, and adding 8mol/L urea solution to 2ml (namely adjusting the protein concentration to 1.0 mg/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), the ultrafiltration tube is sheathed in a 15ml centrifuge tube, and is centrifuged at 4000g and 24 ℃ until the liquid in the upper chamber (the upper chamber is the ultrafiltration tube) in the tube 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, and 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 protein ₄ HCO ₃ Washing and replacing the solution with 50mmol/L NH ₄ HCO ₃ After that, pancreatin was added in a ratio of 1 ₄ HCO ₃ Adding 20. Mu.g of pancreatin and adding 1ml of 50mmol/L NH into the lower chamber ₄ HCO ₃ Incubating for 16h at 37 ℃, replacing a new collecting pipe to collect centrifuged liquid, namely a total peptide fragment mixture, and drying the peptide fragments by using a freeze dryer;

6) Desalting the peptide segment: the resulting dried peptide fragment was dissolved in phase A (1% acetonitrile, 0.1% trifluoroacetic acid solution) and loaded onto an equilibrated 4.6mm C18 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 three times with precooled PBS buffer, 500g,15s;

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 upper part and the lower part, uniformly mixing, centrifuging for 15s at 500g, and washing 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; add 100. Mu.L Elution Buffer (Elution Buffer), reverse up and down for 1min,500g, centrifuge for 15s, repeat twice more, total collection volume is 300. Mu.L;

4) The collected liquid was drained and stored at-20 ℃ to obtain acetylated peptide fragments.

Example 2 LC-MS/MS identification and data banking

LC-MS/MS identification

1) The peptide fragments after enrichment were not fractionated and directly subjected to nano LC-MS/MS analysis, using 0.1% formic acid-acetonitrile (A) and 0.1% formic acid-water (B) as mobile phases, and the peptide fragments of each sample were resuspended in Mass Spectrometry mobile phase A solution, and separated on a C18 column (3 μm,75 μm × 15 cm) at 500nL per minute by an EasyNano LC1000 System (San Jose, thermo Fisher), with flow-through linear gradients as shown in Table 1:

TABLE 1 LC-MS/MS elution conditions

Time/min	Flow rate (nL/min)	A％	B％
				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) Peptide fragments were identified by mass spectrometry 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 350-1550, and the child ion scanning range is as follows: starting at 100 m/z. Other parameters are set as follows: the MS1 and MS2 resolutions are set to 120K and 30K, respectively; automatic Gain Control (AGC) is used, MS1 and MS2 are 1e6 and 1e5, respectively; the ion screening window is a quadrupole rod with the length of 1.6m/z; fragmentation mode: HCD, normalized Collision Energy (NCE) 32, dynamic exclusion time 20s. The secondary maps of peptide segments NLFTQTLGLGSQK (ac) ER and K (ac) HPEPFFALAK in which 55 th and 126 th lysines of SIRT2 are identified by mass spectrum are shown in figure 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

The raw data of mass spectra were pooled by Maxquant (version 1.6.3.3) software, and the protein sequence database was from Uniprot Mus musculus (mouse) and contained 10090 protein sequences. The library search parameters refer to Maxquant instructions:

the sample type is standard, and the multiplex is 1;

the protein digestive enzymes are: trypsin;

fixing and modifying: carbamidomethyl (C);

the variable modification is Acetyl (K), deamidation (N), deamidation (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 peptide fragment used for protein quantification was modified to Acetyl (K) and the peptide fragment was unique.

Example 3 Mass Spectrometry results with increased SIRT2 acetylation levels

In the Maxquant library results table, the intensity value of each site in the Acetyl (K) sites. Txt table was corrected by the total ion current intensity between each sample, and the corrected intensity value was used as the quantification of the acetylated site in each sample. Similarly, each protein intensity value in the protein groups. Txt table for quantification of the non-acetylated peptides was corrected by the total ion current intensity between each sample, and the corrected intensity value was used as a quantitative value for the SIRT2 protein expression level in each sample. The SIRT2 acetylation site intensity difference fold was counted using GraphPad Prism software, as well as protein expression difference fold, and the corresponding p-value was 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 ratio of O to Y of a peptide segment in which acetylated K126 is positioned is 2.03, the value of p is 0.0003, the ratio of O to Y of acetylated K126 modified by protein abundance correction is 1.88, and the value of p is 0.0051, thus confirming that the acetylated modification is not changed due to the change of protein abundance.

In order to further analyze the change of the expression of the SIRT2 protein acetylated K126 in O and Y, xcalibur software (Thermo scientific) is adopted to directly find out an extracted ion flow graph of an SIRT 2K 126 acetylated peptide fragment and a SIRT2 non-acetylated peptide fragment in the original data. Figure 4 shows a peak area plot, peak area statistics, and histogram of SIRT 2K 126 acetylated peptide fragments in each sample, and the results show that K126 acetylation is increased in O relative to Y (p < 0.01) (figure 4). FIG. 5 is a peak plot of the non-acetylated peptide fragment ELYPGQKPTICHYFIR in SIRT2 in each sample, which was statistically analyzed to be not significantly different in O and Y. The above results further demonstrate an increase in acetylation modification of Sirt2 in senescent brain tissue.

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

160. Mu.g of brain protein samples of aged (O) and young (Y) mice were prepared, 20. Mu.L of antibody-coated agarose beads (Pan-acetylated antibody-conjugated resin, pan anti-acetyllysine antibody conjugated agarose beads, PTM BIO, PTM-104) were pipetted with a pipette, washed 3 times with PBS, added with sample protein, and incubated overnight at 4 ℃ together. The following day was taken out of the freezer, centrifuged at 800g for 5 minutes, the supernatant was discarded, the precipitate was washed 5 times with 0.05% PBST, the target protein was eluted with 0.2% formic acid, the supernatant was centrifuged, and after vacuum drying, a 2 Xloading buffer boiling water bath was added for 5 minutes, and Western blot analysis was performed using a primary antibody against SIRT2 (santa cruz, sc-28298) and a secondary antibody against HRP (CST, 7076S) against mouse IgG. The results are shown in figure 6, the SIRT2 protein level in Y and O brain holoprotein is unchanged (Input band), but the expression level of O group of the pan-acetylated antibody enriched band is higher than that of Y group, and as shown by the histogram statistics, the acetylation level of the SIRT2 protein in O group is 2.39 times higher than that of Y group, confirming the increase of SIRT2 acetylation modification level in the brain of the aged mice.

Example 5 SIRT2 conservation assay

The sequence conservation of the mouse, rat, human, cynomolgus monkey, and the T.thuringiensis SIRT2 protein K55 and K126 is analyzed. The sequences of mouse SIRT2 proteins were all drawn from the Uniprot protein database, mouse: q8VDQ8, rat: q5RJQ4, human:

ID Q8IXJ6, cynomolgus monkey: Q4R834, sumendortang chimpanzee: and ID is Q5RBF1. The results are shown in FIG. 7, and the analysis shows that K126 and K55 of SIRT2 are conserved in various mammals.

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> auxiliary hospital of Nantong university

<120> acetylation modified SIRT2 protein marker molecule related to central nervous senescence and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 389

<212> PRT

<213> mouse (Mus musculus)

<220>

<223> amino acid sequence of SIRT2 protein

<400> 1

Met Ala Glu Pro Asp Pro Ser Asp Pro Leu Glu Thr Gln Ala Gly Lys

1 5 10 15

Val Gln Glu Ala Gln Asp Ser Asp Ser Asp Thr Glu Gly Gly Ala Thr

20 25 30

Gly Gly Glu Ala Glu Met Asp Phe Leu Arg Asn Leu Phe Thr Gln Thr

35 40 45

Leu Gly Leu Gly Ser Gln Lys Glu Arg Leu Leu Asp Glu Leu Thr Leu

50 55 60

Glu Gly Val Thr Arg Tyr Met Gln Ser Glu Arg Cys Arg Lys Val Ile

65 70 75 80

Cys Leu Val Gly Ala Gly Ile Ser Thr Ser Ala Gly Ile Pro Asp Phe

85 90 95

Arg Ser Pro Ser Thr Gly Leu Tyr Ala Asn Leu Glu Lys Tyr His Leu

100 105 110

Pro Tyr Pro Glu Ala Ile Phe Glu Ile Ser Tyr Phe Lys Lys His Pro

115 120 125

Glu Pro Phe Phe Ala Leu Ala Lys Glu Leu Tyr Pro Gly Gln Phe Lys

130 135 140

Pro Thr Ile Cys His Tyr Phe Ile Arg Leu Leu Lys Glu Lys Gly Leu

145 150 155 160

Leu Leu Arg Cys Tyr Thr Gln Asn Ile Asp Thr Leu Glu Arg Val Ala

165 170 175

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

180 185 190

Ser His Cys Val Asn Thr Ser Cys Arg Lys Glu Tyr Thr Met Gly Trp

195 200 205

Met Lys Glu Lys Ile Phe Ser Glu Ala Thr Pro Arg Cys Glu Gln Cys

210 215 220

Gln Ser Val Val Lys Pro Asp Ile Val Phe Phe Gly Glu Asn Leu Pro

225 230 235 240

Ser Arg Phe Phe Ser Cys Met Gln Ser Asp Phe Ser Lys Val Asp Leu

245 250 255

Leu Ile Ile Met Gly Thr Ser Leu Gln Val Gln Pro Phe Ala Ser Leu

260 265 270

Ile Ser Lys Ala Pro Leu Ala Thr Pro Arg Leu Leu Ile Asn Lys Glu

275 280 285

Lys Thr Gly Gln Thr Asp Pro Phe Leu Gly Met Met Met Gly Leu Gly

290 295 300

Gly Gly Met Asp Phe Asp Ser Lys Lys Ala Tyr Arg Asp Val Ala Trp

305 310 315 320

Leu Gly Asp Cys Asp Gln Gly Cys Leu Ala Leu Ala Asp Leu Leu Gly

325 330 335

Trp Lys Lys Glu Leu Glu Asp Leu Val Arg Arg Glu His Ala Asn Ile

340 345 350

Asp Ala Gln Ser Gly Ser Gln Ala Pro Asn Pro Ser Thr Thr Ile Ser

355 360 365

Pro Gly Lys Ser Pro Pro Pro Ala Lys Glu Ala Ala Arg Thr Lys Glu

370 375 380

Lys Glu Glu Gln Gln

385

Claims

1. An acetylation modified SIRT2 protein marker molecule related to central nervous senescence, which is characterized in that: the acetylation modified SIRT2 protein marker molecule related to central nervous senescence is SIRT2 protein with 126 th lysine and 55 th lysine being modified by acetylation; the amino acid sequence of the SIRT2 protein is shown in SEQ ID NO. 1.

2. Use of the acetylated modified SIRT2 protein marker molecule of claim 1 in the detection of the degree of senescence in cells, tissues, organs or individuals for non-diagnostic and therapeutic purposes.

3. Use according to claim 2, 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;

(4) Quantitatively analyzing acetylation modification of cells or histones 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.

4. Use according to claim 3, 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;

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

the mice comprise mice and rats;

the monkey is a cynomolgus monkey;

the orangutan is a Sumendortan.

6. Use according to claim 3, characterized in that:

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

7. Use according to claim 6, 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; the peptide fragment used for protein quantification was modified to Acetyl K, and the peptide fragment was unique.