CN112063599A

CN112063599A - Acetylation modified SIRT2 protein marker molecule related to central nervous senescence and application thereof

Info

Publication number: CN112063599A
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: 2020-12-11
Anticipated expiration: 2040-06-01
Also published as: CN112063599B

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 position and the 55 th position. The upregulation of lysine acetylation modification at 55 and 126 positions on the 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, 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 5years 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 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 acetylation modified lysine 126 and lysine 55.

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 use of the above acetylated modified SIRT2 protein marker molecule related to central nervous senescence in the detection of cells, tissues, organs or individual senescence degree; 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 a cell, tissue, organ or individual is evaluated by quantitative analysis 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 a 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) 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 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 × 15cm) at 500nL per minute, and the separation gradient is as follows: 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 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 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 peptide segment used for the quantification of acetylated protein is Acetyl (K), and the peptide segment is 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 central nervous senescence is applied to preparing medicines for preventing and treating senescence-related degenerative diseases.

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 biomarker 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 route diagram of the present invention.

FIG. 2 is a secondary spectrum diagram of peptide segments NLFTQTLGLGSQK(ac) ER and K (ac) HPEPFFALAK of SIRT 2K 55 and K126 identified by mass spectrometry; wherein a is a secondary spectrum of a peptide fragment where K55 is located; b is a secondary spectrum detected by K126 in an O1 sample; c is a secondary spectrum detected by K126 in a 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 the results of mass spectrometric identification; 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 peak area statistics diagram of a peptide segment K (ac) HPEPFFALAK in which SIRT 2K 126 is identified by mass spectrometry in each sample; wherein a is the ion flow pattern of peptide segment K (ac) HPEPFFALAK in each sample; b is the corresponding peak area of peptide fragment k (ac) HPEPFFALAK in each sample; c is a histogram of statistical results of the b picture.

FIG. 5 is an ion flow graph and peak area statistics graph of a mass-spectrometrically identified SIRT2 nonacetylated peptide fragment ELYPGQFKPTICHYFIR in each sample; where) is the ion flow map of peptide segment ELYPGQFKPTICHYFIR in each sample; b is the corresponding peak area of 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 results 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 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 into 8mol/L urea buffer solution according to the concentration of 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, and carrying out 5 cycles at the interval of 10s 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 and old mice, and adding 8mol/L urea solution to 2ml (namely adjusting the protein concentration to 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 (the upper chamber is the ultrafiltration tube) in the tube is less than 100 mu l;

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 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 original protein)₄HCO₃Adding pancreatin 20 μ g, adding NH 50 mmol/L1 ml in the lower chamber₄HCO₃Incubating at 37 deg.C for 16h, collecting centrifuged liquid by a new collecting tube to obtain total peptide mixture, and freeze-drying the peptide mixture with a freeze dryerPumping to dry;

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 4.6mm 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, 15 s;

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), inverting for 1min, 500g, centrifuging for 15s, repeating twice more, 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 nano LC-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, separated by an EasyNano LC1000 system (San Jose, Thermo Fisher) on a C18 column (3 μm, 75 μm × 15cm) at 500nL per minute, and the flow-through linear gradients are 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) The peptide fragments were mass-characterized by means of DDA using an Orbitrap Fusion Lumos (Bremen, Thermo Fisher) mass spectrometer. 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, dynamic exclusion time 20 s. The secondary maps of peptide portions NLFTQTLGLGSQK(ac) ER and K (ac) HPEPFFALAK of 55 th lysine and 126 th lysine of SIRT2 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

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: 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 modification of the peptide fragment for protein quantification is Acetyl (K), and the peptide fragment is unique.

Example 3 Mass Spectrometry results with increased levels of SIRT2 acetylation

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 for quantification of non-acetylated peptide fragments was corrected for total ion flux intensity between each sample, and the corrected intensity value was used as a quantitative value for the amount of SIRT2 protein expression in each sample. The intensity difference of SIRT2 acetylation site is counted by using GraphPad Prism software, and protein expression difference is counted, and 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 ratio of O to Y of the peptide segment in which acetylated K126 is located is 2.03, the p value is 0.0003, the ratio of O to Y of K126 acetylated modified after protein abundance correction is 1.88, and the p value is 0.0051, which proves 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 diagram of an SIRT 2K 126 acetylated peptide segment and a SIRT2 non-acetylated peptide segment in 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 indicate that K126 acetylation is increased in O relative to Y (p <0.01) (figure 4). Figure 5 is a peak plot of the non-acetylated peptide fragment ELYPGQFKPTICHYFIR 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 next day, the cells were removed from 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 the supernatant was removed after vacuum drying and subjected to Western blot analysis using a 2 Xloading buffer in a boiling water bath for 5 minutes, and a primary antibody against SIRT2 (santa cruz, sc-28298) and a secondary antibody against mouse IgG (HRP CST, 7076S) against mouse IgG. The results are shown in figure 6, the levels of SIRT2 protein in Y and O brain holoprotein are 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 statistical results, the acetylation level of SIRT2 protein in O group is 2.39 times higher than that of Y group, which confirms the increase of SIRT2 acetylation modification level in the brain of aged mice.

Example 5 SIRT2 conservation assay

Mouse, rat, human, cynomolgus, sumatrio chimpanzee SIRT2 proteins K55 and K126 were sequence conserved. The sequences of the mouse SIRT2 proteins were all drawn from the Uniprot protein database, mouse: q8VDQ8, rat: q5RJQ4, human:

ID Q8IXJ6, cynomolgus monkey: Q4R834, Sumendortang chimpanzee: ID Q5RBF 1. 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> affiliated 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.

2. The SIRT2 protein marker molecule with acetylation modification related to central nervous senescence of claim 1, wherein: the amino acid sequence of the SIRT2 protein is shown in SEQ ID NO. 1.

3. Use of the acetylation modified SIRT2 protein marker molecule related to central nervous system aging of claim 1 or 2 in the detection of the aging degree of 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;

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:

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

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; the peptide fragment used for protein quantification was modified to Acetyl K, and the peptide fragment was unique.

9. Use of the acetylated modified SIRT2 protein marker molecule related to central nervous senescence of claim 1 or 2 in the preparation of a kit for predicting senescence-related degenerative disease.

10. Use of the acetylation modified SIRT2 protein marker molecule related to central nervous senescence of claim 1 or 2 in the preparation of drugs as drug targets in preventing and/or treating senescence-related degenerative diseases.