CN113186182A - CAH2 protein as skin aging protein marker and its non-invasive extraction method - Google Patents
CAH2 protein as skin aging protein marker and its non-invasive extraction method Download PDFInfo
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- CN113186182A CN113186182A CN202110419795.XA CN202110419795A CN113186182A CN 113186182 A CN113186182 A CN 113186182A CN 202110419795 A CN202110419795 A CN 202110419795A CN 113186182 A CN113186182 A CN 113186182A
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention provides a skin aging protein marker-CAH 2 protein (carbonic anhydrase 2) and a noninvasive extraction method thereof. According to the invention, the carbonic anhydrase 2 content in the skin of people of all ages is detected, and the carbonic anhydrase 2 expression is found to be up-regulated with the increase of the ages, so that the skin aging degree is judged in an auxiliary manner, the skin aging is intervened in advance before the appearance of the skin aging, and the aging is effectively delayed. The method is simple and easy to implement, and has wide market prospect.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a skin aging protein marker CAH2 protein (carbonic anhydrase 2) and a noninvasive extraction method thereof.
Background
As the human body ages, the skin is aged, the skin aging is serious, the human body is very old, and anyone is reluctant to see that the human body is more old than the aged person, so the beauty treatment or medical beauty treatment is more and more concerned by people. The correct judgment of the aging degree of the skin and the judgment of whether the physiological age of skin aging coincides with the actual age are the prerequisites of beauty treatment or medical beauty treatment. Cosmetic or medical cosmetology can only be oriented if an inherent source of aging leading to accelerated skin aging is found.
Among human and animal blood, carbonic anhydrase is one of the major protein components in erythrocytes, and has a secondary importance and content to hemoglobin, and is a zinc protein, present in vertebrate erythrocytes and in various tissues of many animals and in plant leaves. It has a rapid conversion effect on carbonate and bicarbonate ions in erythrocytes. It acts on the secretion of hydrochloric acid in the stomach and, in general, has the effect of regulating the pH of body fluids. It is also considered to be involved in photosynthesis of plants.
CAH2 Carbonic Anhydrase (CA) is a ubiquitously expressed enzyme that reversibly hydrates and hydrates carbon dioxide to bicarbonate and protons. CHA2 is almost completely expressed in differentiated keratinocytes. The enzyme is involved in maintaining the pH of the cells, water transport and ion homeostasis. Many skin diseases are associated with the destruction of the skin's microbial environment. For example, staphylococcus aureus prefers a neutral pH. The disturbance of the pH of the skin surface may lead to microbial colonization and infection of the skin, thereby promoting the development of inflammatory skin diseases.
At present, carbonic anhydrase is not used as a precedent for assisting in judging the degree of skin aging.
Disclosure of Invention
The invention aims to provide application of carbonic anhydrase 2 (the amino acid sequence of which is shown in SEQ ID NO. 1) in auxiliary judgment of aging degree.
The invention provides the application of a substance for detecting the content of carbonic anhydrase 2 in preparing products; the product has the function of assisting in judging the aging degree.
The invention provides the application of a substance for detecting the content of carbonic anhydrase 2 in preparing products; the product has the function of assisting in judging the skin aging degree.
The substance for detecting the content of carbonic anhydrase 2 can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; and more particularly to an orbitrap high resolution mass spectrometer.
The substance for detecting the carbonic anhydrase 2 content also includes a substance used for sampling from the skin surface. Specifically, the adhesive tape may be a 3M adhesive.
In any of the above applications, the product is a kit or a chip.
The invention also protects a product comprising a substance for detecting the carbonic anhydrase 2 content; the function of the product is as follows (a) or (b):
(a) the aging degree is judged in an auxiliary manner;
(b) and (4) assisting in judging the skin aging degree.
In the product, the substance for detecting the content of carbonic anhydrase 2 can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; and more particularly to an orbitrap high resolution mass spectrometer.
In the product, the substance for detecting the content of carbonic anhydrase 2 further comprises a substance for sampling from the skin surface. Specifically, the adhesive tape may be a 3M adhesive.
The product also comprises a carrier which is recorded with the following detection methods:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of carbonic anhydrase 2 in the obtained skin sample of the subject;
3) comparing the carbonic anhydrase 2 content measured in the step 2) with the carbonic anhydrase 2 content value in the skin of the person with normal aging at the age group, and judging the skin aging degree of the subject according to the comparison result.
And further comprising 4) comparing the carbonic anhydrase 2 content measured in the step 2) with standard curve of carbonic anhydrase 2 content in skin of normally aging person of each age, judging physiological age of skin of the subject according to the comparison result,
if the skin aging degree of the subject is faster or the physiological age of the skin of the subject is larger than the actual age of the subject, corresponding intervention measures are adopted to delay the skin aging and achieve the beautifying effect.
The invention also provides a system for assisting in judging the aging degree.
The system for assisting in judging the aging degree provided by the invention can comprise the following modules:
(1) a data receiving module; the data receiving module is configured to receive data on the content of carbonic anhydrase 2 in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store carbonic anhydrase 2 content data in the skin of a normally aging person consistent with the age group of the subject or the data storage module is configured to store a standard curve of carbonic anhydrase 2 content in the skin of a normally aging person at each age group;
(3) a data comparison module: the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with the carbonic anhydrase 2 content data stored in the data storage module in the skin of the person with normal aging consistent with the age bracket of the subject; or the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with standard curves of the carbonic anhydrase 2 content in the skin of the normally aging person at 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 larger than the actual age of the subject, and output the judgment result.
The system for assisting in judging the aging degree further comprises an instrument for detecting the content of carbonic anhydrase 2 in the skin sample of the subject, wherein the instrument can be specifically an orbital trap high-resolution mass spectrometer;
the system for assisting in determining the degree of aging may further include a substance for obtaining a sample of the skin of the epidermis of the subject. In particular, the substance is a sticker, more particularly, the sticker may be a 3M glue.
The method for assisting in judging the skin aging degree provided by the invention comprises the following steps:
1) sticking the skin surface with an adhesive plaster, and then tearing off to obtain a skin sample of the epidermis of the subject; the method comprises the following steps of sticking a 3M adhesive on the curved side part of the forearm of a subject, and slightly tearing off the adhesive to ensure that the skin of the subject is not damaged and only a falling layer on the surface of the skin of the subject is stained;
2) detecting the content of carbonic anhydrase 2 in the obtained skin sample of the subject;
3) comparing the carbonic anhydrase 2 content measured in the step 2) with the carbonic anhydrase 2 content value in normal human skin with the same age of the subject, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the carbonic anhydrase 2 content measured in the step 2) with standard curves of the carbonic anhydrase 2 content in normal human skin of each age, and judging the physiological age of the skin of the subject according to the comparison result;
judging that the physiological age of the skin of the subject is older than the actual age when the content of the carbonic anhydrase 2 in the skin of the subject is higher than that in the skin of a normal person; the physiological age of the skin of the subject is judged to be younger than the actual age, with the content of carbonic anhydrase 2 in the skin of the subject being lower than the content of carbonic anhydrase 2 in the skin of a normal human.
The invention also provides a method for non-invasively obtaining carbonic anhydrase 2 from skin, comprising: (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 invention further provides a method for determining the relative content of carbonic anhydrase 2 in an epidermal skin sample based on mass spectrum, which 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; separating by UHPLC; 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.
According to the invention, the carbonic anhydrase 2 content in the skin of people of all ages is detected, and the carbonic anhydrase 2 expression is found to be up-regulated with the increase of the ages, so that the skin aging degree is judged in an auxiliary manner, the skin aging is intervened in advance before the appearance of the skin aging, and the aging is effectively delayed. The method is simple and easy to implement, and has wide market prospect.
Drawings
FIG. 1 is a mass spectrum of a characteristic peptide fragment (YDPSLKPLSVSYDQATSLR) of carbonic anhydrase 2 detected.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
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.
The invention provides application of carbonic anhydrase 2 in auxiliary judgment of aging degree.
The invention provides the application of a substance for detecting the content of carbonic anhydrase 2 in preparing products; the product has the function of assisting in judging the aging degree.
The invention provides the application of a substance for detecting the content of carbonic anhydrase 2 in preparing products; the product has the function of assisting in judging the skin aging degree.
The substance for detecting the content of carbonic anhydrase 2 can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; and more particularly to an orbitrap high resolution mass spectrometer.
The substance for detecting the carbonic anhydrase 2 content also includes a substance used for sampling from the skin surface.
In any of the above applications, the product is a kit or a chip.
The invention also protects a product comprising a substance for detecting the carbonic anhydrase 2 content; the function of the product is as follows (a) or (b):
(a) the aging degree is judged in an auxiliary manner;
(b) and (4) assisting in judging the skin aging degree.
In the product, the substance for detecting the content of carbonic anhydrase 2 can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; and more particularly to an orbitrap high resolution mass spectrometer.
In the product, the substance for detecting the content of carbonic anhydrase 2 further comprises a substance for sampling from the skin surface.
The product also comprises a carrier which is recorded with the following detection methods:
1) taking a skin sample of a subject;
2) detecting the content of carbonic anhydrase 2 in the obtained skin sample of the subject;
3) comparing the carbonic anhydrase 2 content measured in the step 2) with the carbonic anhydrase 2 content value in the skin of the person with normal aging at the age group, and assisting in judging the skin aging degree of the subject according to the comparison result.
And further comprising 4) comparing the carbonic anhydrase 2 content measured in the step 2) with standard curve of carbonic anhydrase 2 content in skin of normally aging person of each age, judging physiological age of skin of the subject according to the comparison result,
if the skin aging degree of the subject is faster or the physiological age of the skin of the subject is larger than the actual age of the subject, corresponding intervention measures are adopted to delay the skin aging and achieve the beautifying effect.
The invention also provides a system for assisting in judging the aging degree.
The system for assisting in judging the aging degree provided by the invention can comprise the following modules:
(1) a data receiving module; the data receiving module is configured to receive data on the content of carbonic anhydrase 2 in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store carbonic anhydrase 2 content data in the skin of a normally aging person consistent with the age group of the subject or the data storage module is configured to store a standard curve of carbonic anhydrase 2 content in the skin of a normally aging person at each age group;
(3) a data comparison module: the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with the carbonic anhydrase 2 content data stored in the data storage module in the skin of the person with normal aging consistent with the age bracket of the subject; or the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with standard curves of the carbonic anhydrase 2 content in the skin of the normally aging person at 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 larger than the actual age of the subject, and output the judgment result.
The system for assisting in judging the aging degree further comprises an instrument for detecting the content of carbonic anhydrase 2 in the skin sample of the subject, wherein the instrument can be specifically an orbital trap high-resolution mass spectrometer;
the system for assisting in determining the degree of aging may further comprise a substance for obtaining a sample of the skin of the subject.
Examples
Taking a crowd with a relatively close living environment and a healthy body as a sample, and carrying out sampling on the skin sample of the forearm curved side part (sampling by using 3M sticky skin) by a noninvasive method.
The method for measuring the content of different proteins in the skin sample of the subject comprises the following steps:
(1) high performance liquid phase
Obtaining a sample: the 3M medical adhesive plaster is stuck to the curved side part of the forearm, and photoaging factors are eliminated. (3M medical adhesive is 2.4cm, and the manufacturer: 3M Health Care 3M Brookings.) and slightly tearing off the 3M adhesive after 1 minute to ensure that the skin of the subject is not damaged and obtain a banded skin sample;
(1) tape-like skin samples were cut into small pieces (0.5x 0.5 cm) and deposited on a glass plate with a sterile razor blade before being transferred to a 1.5 ml centrifuge tube; (2) an appropriate amount of lysis buffer sample without SDS was added, 2mM EDTA, 1XCocktail was added, and then placed on ice for 5 minutes. Then, 10mM DTT was added and the sample was soaked overnight. (3) centrifuging the supernatant at 25,000g at 4 ℃ for 15 minutes, recovering the supernatant and treating the DTT with 10mM for 1 hour in a water bath at 56 ℃; (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 obtain the final protein solution supernatant. Protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE (including whether protein extraction was sufficient, presence or absence of degradation, and quantitative estimation). For proteolysis, 100. mu.g of protein per sample was taken, trypsin was added and the hydrolysis was carried out for 4 hours at 37 ℃ (protein: enzyme ratio 40: 1). Subsequently trypsin was added again in the same ratio for 8 hours at 37 ℃. Desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample;
the dried peptide fragment sample was reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000g for 10 min, and the supernatant was injected. The separation was carried out by the Thermo company UltiMate 3000 UHPLC. The sample was first enriched and desalted in a trap column and then connected in series with a self-contained C18 column (150 μm internal diameter, 1.8 μm column size, 25cm column length) at a flow rate of 500nl/min with 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 ℃ C. for 180min, 5% mobile phase B. The end of the nanoliter liquid phase separation was directly connected to the mass spectrometer.
(2) DDA mass spectrometric detection
The liquid phase separated peptide fragments were ionized by a nanoESI source and then introduced into a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DDA (data-dependent acquisition) mode 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: charge 2+ to 7+, parent ion with a peak intensity above 10,000 ranked first 20. The ion fragmentation mode was HCD and fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s. The AGC is set as: primary 3E6, secondary 1E 5.
(3) DIA mass spectrometric detection
The liquid phase separated peptide fragments were ionized by nanoESI source and then introduced into a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DIA (data-independent acquisition) mode 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; the 350-1500Da fragment is divided into 40 windows for fragmentation and signal collection. The ion fragmentation mode was HCD and fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s. The AGC is set as: primary 3E6, secondary 1E 5.
FIG. 1 is a mass spectrum of a characteristic peptide fragment (YDPSLKPLSVSYDQATSLR) of carbonic anhydrase 2 detected.
The data for the relative content of carbonic anhydrase 2 in the skin samples of the subjects are as follows:
| numbering | Age (age) | Relative content of carbonic anhydrase 2 |
| 1 | 24y | 11.13852583 |
| 2 | 55y | 11.55075645 |
| 3 | 63y | 11.76089485 |
| 4 | 65y | 12.52744995 |
| 5 | 72y | 12.91279426 |
As can be seen from the data in the above table, the relative level of carbonic anhydrase 2 in the skin sample of the subject increases with age.
In practical application, firstly, the skin of each normal age person with statistical significance is collected as a sample, the relative content of carbonic anhydrase 2 in each skin sample is respectively measured, an age-carbonic anhydrase 2 relative content standard curve is drawn, then the relative content of carbonic anhydrase 2 in the skin sample of a subject is measured, and the skin aging degree of the subject is judged by comparing the relative content of carbonic anhydrase 2 in the skin sample of the subject with the standard curve. Or to a group of people of a certain age. For example, to serve a 60 year old person in a city, a statistical skin sample of a 60 year old normal person living in the city is first collected, the relative content of carbonic anhydrase 2 in each skin sample is determined, and the 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 carbonic anhydrase 2 in the skin of the subject is measured by the same method as the threshold value, and when the content of carbonic anhydrase 2 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 carbonic anhydrase 2 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 carbonic anhydrase 2 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 the present example, is possible and should be protected by the present invention.
Besides the skin, the content of carbonic anhydrase 2 can also be used as an index for assisting in the judgment of the overall aging degree of a human.
Gene carbonic anhydrase 2, the amino acid sequence of which is shown in SEQ ID NO.1, is as follows:
MSHHWGYGKHNGPEHWHKDFPIAKGERQSPVDIDTHTAKYDPSLKPLSVSYD QATSLRILNNGHAFNVEFDDSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEHT VDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDV LDSIKTKGKSADFTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSE QVLKFRKLNFNGEGEPEELMVDNWRPAQPLKNRQIKASFK 。
SEQUENCE LISTING
<110> Yangsen
<120> a skin aging protein marker-CAH 2 protein and its non-invasive extraction method
<130> GNCAQ211030
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 260
<212> PRT
<213> Homo sapiens
<400> 1
Met Ser His His Trp Gly Tyr Gly Lys His Asn Gly Pro Glu His Trp
1 5 10 15
His Lys Asp Phe Pro Ile Ala Lys Gly Glu Arg Gln Ser Pro Val Asp
20 25 30
Ile Asp Thr His Thr Ala Lys Tyr Asp Pro Ser Leu Lys Pro Leu Ser
35 40 45
Val Ser Tyr Asp Gln Ala Thr Ser Leu Arg Ile Leu Asn Asn Gly His
50 55 60
Ala Phe Asn Val Glu Phe Asp Asp Ser Gln Asp Lys Ala Val Leu Lys
65 70 75 80
Gly Gly Pro Leu Asp Gly Thr Tyr Arg Leu Ile Gln Phe His Phe His
85 90 95
Trp Gly Ser Leu Asp Gly Gln Gly Ser Glu His Thr Val Asp Lys Lys
100 105 110
Lys Tyr Ala Ala Glu Leu His Leu Val His Trp Asn Thr Lys Tyr Gly
115 120 125
Asp Phe Gly Lys Ala Val Gln Gln Pro Asp Gly Leu Ala Val Leu Gly
130 135 140
Ile Phe Leu Lys Val Gly Ser Ala Lys Pro Gly Leu Gln Lys Val Val
145 150 155 160
Asp Val Leu Asp Ser Ile Lys Thr Lys Gly Lys Ser Ala Asp Phe Thr
165 170 175
Asn Phe Asp Pro Arg Gly Leu Leu Pro Glu Ser Leu Asp Tyr Trp Thr
180 185 190
Tyr Pro Gly Ser Leu Thr Thr Pro Pro Leu Leu Glu Cys Val Thr Trp
195 200 205
Ile Val Leu Lys Glu Pro Ile Ser Val Ser Ser Glu Gln Val Leu Lys
210 215 220
Phe Arg Lys Leu Asn Phe Asn Gly Glu Gly Glu Pro Glu Glu Leu Met
225 230 235 240
Val Asp Asn Trp Arg Pro Ala Gln Pro Leu Lys Asn Arg Gln Ile Lys
245 250 255
Ala Ser Phe Lys
260
Claims (10)
1. The application of the substance for detecting the content of carbonic anhydrase 2 in the preparation of products; the product has the function of assisting in judging the aging degree.
2. The application of the substance for detecting the content of carbonic anhydrase 2 in the preparation of products; the product has the function of assisting in judging the skin aging degree.
3. A product comprising a substance for detecting the level of carbonic anhydrase 2; the function of the product is as follows (a) or (b):
(a) the aging degree is judged in an auxiliary manner;
(b) and (4) assisting in judging the skin aging degree.
4. The product of claim 3, wherein: in the product, the substance for detecting the content of carbonic anhydrase 2 is a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; and/or the substance for detecting the content of carbonic anhydrase 2 is an orbital trap high resolution mass spectrometer.
5. The product of claim 4, wherein: the product also comprises a carrier which is recorded with the following detection methods:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of carbonic anhydrase 2 in the obtained skin sample of the subject;
3) comparing the carbonic anhydrase 2 content measured in the step 2) with the carbonic anhydrase 2 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 carbonic anhydrase 2 content measured in the step 2) with a standard curve of the carbonic anhydrase 2 content in the skin of the normally aging person of each age group, and judging the physiological age of the skin of the subject according to the comparison result.
6. A system for assisting in determining the degree of aging, comprising the following modules:
(1) a data receiving module; the data receiving module is configured to receive data on the content of carbonic anhydrase 2 in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store carbonic anhydrase 2 content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store a standard curve of carbonic anhydrase 2 content in normal human skin at each age group;
(3) a data comparison module: the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with the carbonic anhydrase 2 content data stored in the data storage module in normal human skin consistent with the age bracket of the subject; or the data comparison module is configured to compare the carbonic anhydrase 2 content data received by the data receiving module from the skin sample of the subject with the standard curve of the carbonic anhydrase 2 content in 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.
7. The system of claim 6, wherein: the system for assisting in judging the aging degree also comprises a substance for detecting the content of carbonic anhydrase 2 in the skin sample of the subject; specifically, the substance for detecting the content of carbonic anhydrase 2 in the skin sample of the subject is a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof; the instrument for detecting the content of carbonic anhydrase 2 in the skin sample of the subject is an orbital trap high-resolution mass spectrometer.
8. A method for assisting in judging the degree of skin aging, comprising the steps of:
1) attaching the skin surface with adhesive, and tearing off to obtain skin sample of the epidermis of the subject;
2) detecting the content of carbonic anhydrase 2 in the obtained skin sample of the subject;
3) comparing the carbonic anhydrase 2 content measured in the step 2) with the carbonic anhydrase 2 content value in normal human skin with the same age of the subject, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the carbonic anhydrase 2 content measured in the step 2) with standard curves of the carbonic anhydrase 2 content in normal human skin of each age, and judging the physiological age of the skin of the subject according to the comparison result;
determining that the physiological age of the skin of the subject is younger than the actual age when the content of the carbonic anhydrase 2 in the skin of the subject is lower than the content of the carbonic anhydrase 2 in the skin of a normal person; the content of the carbonic anhydrase 2 in the skin of the subject is higher than that of the carbonic anhydrase 2 in the skin of a normal person, and the physiological age of the skin of the subject is judged to be older than the actual age.
9. A method of non-invasively obtaining carbonic anhydrase 2 from skin comprising: (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.
10. The method for measuring the relative content of carbonic anhydrase 2 in the 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; separating by UHPLC; 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.
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