CN113651878A - Skin aging protein marker-1433T protein and non-invasive extraction method thereof - Google Patents
Skin aging protein marker-1433T protein and non-invasive extraction method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
Abstract
The invention discloses a skin aging protein marker-1433T protein and a non-invasive extraction method thereof. The application of the 1433T protein in the auxiliary judgment of the aging degree and the extraction and detection method are disclosed, aiming at finding out the intrinsic factors causing skin aging from the root, intervening the skin aging in advance before the appearance of the skin aging, and in addition, correctly judging the aging degree of the skin and judging whether the physiological age of the aging accords with the actual age, and providing reference and direction for beauty treatment or medical beauty treatment.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a skin aging protein marker-1433T protein and a noninvasive extraction method thereof.
Background
Skin aging, also known as skin aging, refers to the functional aging damage of skin, which reduces the protection and regulation ability of skin to human body, so that the skin can not adapt to the change of internal and external environment, and the change of the overall appearance such as color, luster, shape, texture, etc. Aging of the skin is classified into intrinsic aging and extrinsic aging. Endogenous aging refers to the natural aging of the skin as it ages. It is manifested as whitening of the skin, fine wrinkles, decreased elasticity, skin laxity, etc. The most prominent cause of extrinsic aging is photoaging due to sun exposure. Skin discoloration manifested as wrinkles, loose skin, roughness, yellowish or grayish yellow, telangiectasia, formation of pigmented spots, and the like.
With the improvement of living standard of people, people pay more and more attention to skin care, but usually only pay attention to the external expression of skin aging, such as wrinkle, color spot, pore thickness degree and other information, and judge the aging degree of skin, the method for judging the aging degree of skin cannot find out the internal factors causing skin aging from the root, cannot intervene skin aging in advance before the external expression of skin aging occurs, and in addition, correctly judge the aging degree of skin and judge whether the physiological age of aging accords with the actual age, and is also the precondition of cosmetology or medical cosmetology.
The 1433T protein is associated with "senescence". The 14-3-3 proteins are a highly conserved class of proteins that are widely present in different eukaryotic cells. The 14-3-3 protein has multiple isoforms, e.g., β, ε, η, γ, τ, σ, ζ, with different expression levels in different tissues. It antagonizes growth factor signaling on the inner nuclear membrane, which is an important driver of pathological fibrosis in multiple organ systems.
There is currently no precedent for using the 1433T protein (containing 3 LEM domains) as an aid in determining the degree of skin aging.
Disclosure of Invention
A method for non-invasive extraction of 1433T protein in skin, the method comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
Preferably, the method for determining the relative content of 1433T protein in an epidermal skin sample based on mass spectrometry comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample;
(3) detection of
Redissolving the dried peptide fragment sample with mobile phase A (2% ACN, 0.1% FA), centrifuging at 20,000g for 10 min, and sampling the supernatant; separation was performed by U1433T protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
Preferably, the substance for detecting 1433T protein content is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the content of 1433T protein is an orbital trap high-resolution mass spectrometer.
Preferably, the P-value of the 1433T protein is 0.013267471.
Preferably, the method for judging the aging degree and the skin aging degree by the 1433T protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of 1433T protein in the skin sample of the obtained subject;
3) comparing the 1433T protein content measured in the step 2) with the 1433T protein content value in the skin of the person with normal aging of the age group, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the 1433T protein content measured in the step 2) with a 1433T protein content standard curve in the skin of a normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.
Preferably, the system for assisting in determining the degree of aging comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive 1433T protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store 1433T protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store 1433T protein content standard curves in normal human skin of each age group;
(3) a data comparison module: the data comparison module is configured to compare the 1433T protein content data of the skin sample of the subject received by the data receiving module with the 1433T protein content data of the normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the 1433T protein content data in the skin sample of the subject received by the data receiving module with the 1433T protein content standard curve in the normal human skin of each age group stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
The invention detects the 1433T protein content in the skin, assists in judging the skin aging degree, finds out the intrinsic factor causing the skin aging from the root, intervenes the skin aging in advance before the appearance of the skin aging, in addition, can correctly judge the skin aging degree and judge whether the aging physiological age is consistent with the actual age, and provides reference and direction for beauty treatment or medical beauty treatment.
Drawings
FIG. 1 is a mass spectrum of the characteristic peptide fragment (QTIDNSQGAYQEAFDISKK) of the (1433T protein) obtained by detection.
Detailed Description
The present invention is further described below by way of specific examples, but the present invention is not limited to only the following examples. Variations, combinations, or substitutions of the invention, which are within the scope of the invention or the spirit, scope of the invention, will be apparent to those of skill in the art and are within the scope of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
A method for non-invasive extraction of 1433T protein in skin, the method comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
The method for measuring the relative content of 1433T protein in an epidermal skin sample based on mass spectrum comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample;
(3) detection of
Redissolving the dried peptide fragment sample with mobile phase A (2% ACN, 0.1% FA), centrifuging at 20,000g for 10 min, and sampling the supernatant; separation was performed by U1433T protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
The substance for detecting the content of the 1433T protein is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the content of 1433T protein is an orbital trap high-resolution mass spectrometer.
The P-value of the 1433T protein was 0.013267471.
The method for judging the aging degree and the skin aging degree by the 1433T protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of 1433T protein in the skin sample of the obtained subject;
3) comparing the 1433T protein content measured in the step 2) with the 1433T protein content value in the skin of the person with normal aging of the age group, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the 1433T protein content measured in the step 2) with a 1433T protein content standard curve in the skin of a normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.
The system for assisting in judging the aging degree comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive 1433T protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store 1433T protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store 1433T protein content standard curves in normal human skin of each age group;
(3) a data comparison module: the data comparison module is configured to compare the 1433T protein content data of the skin sample of the subject received by the data receiving module with the 1433T protein content data of the normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the 1433T protein content data in the skin sample of the subject received by the data receiving module with the 1433T protein content standard curve in the normal human skin of each age group stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
FIG. 1 is a mass spectrum of a characteristic peptide fragment (QTIDNSQGAYQEAFDISKK) of 1433T protein obtained by detection.
Randomly sampling 11 women and 9 men of normal healthy Chinese as subjects, wherein the relative content data of 1433T protein in skin samples are as follows:
| group A young group (number) | Age (age) | Relative content of 1433T protein |
| 1 | 20y (Man) | 10.67181908 |
| 2 | 26y (woman) | 9.657305524 |
| 3 | 26y (woman) | 10.48959667 |
| 4 | 27y (woman) | 13.72267607 |
| 5 | 31y (Man) | 11.81857223 |
| Group B youth group (number) | Age (age) | Relative content of 1433T protein |
| 1 | 52y (Man) | 9.162491194 |
| 2 | 55y (Man) | 14.93530626 |
| 3 | 63y (woman) | 9.96036855 |
| 4 | 65y (woman) | 10.79832702 |
| 5 | 65y (Man) | 11.68682307 |
| 6 | 72y (woman) | 11.18122914 |
As can be seen from the data in the above table, the relative content of 1433T protein in skin samples of subjects increased with age.
In practical application, firstly, the skin of each statistically significant normal person of each age is collected as a sample, the relative content of 1433T protein in each skin sample is respectively measured, for example, to serve a group of people 35 years old in a certain city, then firstly, the skin sample of the statistically significant normal person 35 years old living in the city is collected, the relative content of 1433T protein in each skin sample is measured, and an average value is obtained. The average value is a threshold value for measuring the skin aging degree of the subject, when the subject is evaluated, the content of 1433T protein in the skin is measured by the same method as the threshold value, and when the content of 1433T protein is lower than the threshold value, the physiological age of the skin of the subject is younger than the actual age; and when the content of the 1433T protein in the skin of the subject is higher than the threshold value, judging that the physiological age of the skin of the subject is older than the actual age.
As to how to measure the content of 1433T protein in the skin, any method capable of determining the absolute and relative content of protein, such as antigen-antibody binding method, etc., other than the method of mass spectrometry in this example, is possible and should be protected by the present invention.
Besides skin, the content of 1433T protein can also be used as an index for assisting in judging the overall aging degree of a human body.
Gene:1433T
Protein 1433T Protein
MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVISSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGDYFRYLA
EVACGDDRKQTIDNSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPELACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN。
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for non-invasive extraction of 1433T protein in skin, the method comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
2. The method of claim 1, wherein: the method for measuring the relative content of 1433T protein in an epidermal skin sample based on mass spectrum comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample;
(3) detection of
Redissolving the dried peptide fragment sample with mobile phase A (2% ACN, 0.1% FA), centrifuging at 20,000g for 10 min, and sampling the supernatant; separation was performed by U1433T protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
3. The 1433T protein assay of claim 2, wherein: the substance for detecting the content of the 1433T protein is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the content of 1433T protein is an orbital trap high-resolution mass spectrometer.
4. The 1433T protein assay of claim 2, wherein: the P-value of the 1433T protein was 0.013267471.
5. The 1433T protein of claim 1, wherein: the method for judging the aging degree and the skin aging degree by the 1433T protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of 1433T protein in the skin sample of the obtained subject;
3) comparing the 1433T protein content measured in the step 2) with the 1433T protein content value in the skin of the person with normal aging of the age group, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the 1433T protein content measured in the step 2) with a 1433T protein content standard curve in the skin of a normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.
6. The method of claim 5, wherein: the system for assisting in judging the aging degree comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive 1433T protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store 1433T protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store 1433T protein content standard curves in normal human skin of each age group;
(3) a data comparison module: the data comparison module is configured to compare the 1433T protein content data of the skin sample of the subject received by the data receiving module with the 1433T protein content data of the normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the 1433T protein content data in the skin sample of the subject received by the data receiving module with the 1433T protein content standard curve in the normal human skin of each age group stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
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| CN114107420A (en) * | 2021-12-23 | 2022-03-01 | 杨森 | Skin aging protein marker BRCC3 protein and noninvasive extraction method thereof |
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