CN113203862A - Skin rejuvenation protein marker-BAF protein and noninvasive extraction method thereof - Google Patents
Skin rejuvenation protein marker-BAF protein and noninvasive extraction method thereof Download PDFInfo
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Abstract
The invention provides a skin rejuvenation protein marker-BAF protein and a noninvasive extraction method thereof. According to the invention, the content of the self-integration obstacle factor (BANF-1) in the skin of each age is detected, and the expression of the self-integration obstacle factor (BANF-1) is found to be reduced with the increase of the age, 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 rejuvenation protein marker-BAF protein 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.
Relevant studies indicate that the factor for the disorder of self-integration (BANF-1, BAF) may be involved in the development of abnormal thymus in patients with Myasthenia Gravis (MG), and in particular in MG thymus atrophy.
Such proteins are associated with the ability to modulate oxidative stress at the DNA level; the DNA repair capacity of human cells decreases with age, and BANF1 controls the DNA damage response to oxidative stress by modulating poly [ ADP-ribose ] polymerase 1(PARP 1). (12 months 2019, PMID: 31796734). Whereas homozygous mutation of BANF1 (c.34G > A [ p.Ala12Thr ] results in premature senility syndrome.
At present, no precedent exists for using the self-integration obstacle factor (BANF-1, BAF) for assisting in judging the aging degree of the skin.
Disclosure of Invention
The invention aims to provide application of a self-integration obstacle factor (BANF-1) (the amino acid sequence of the BANF-1 is shown in SEQ ID NO. 1) in auxiliary judgment of the aging degree.
The invention provides an application of a substance for detecting the content of a self-integration obstacle factor (BANF-1) in preparing a product; the product has the function of assisting in judging the aging degree.
The invention provides an application of a substance for detecting the content of a self-integration obstacle factor (BANF-1) in preparing a product; the product has the function of assisting in judging the skin aging degree.
The substance for detecting the content of the self-integration obstacle factor (BANF-1) 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 content of the self-integration barrier factor (BANF-1) also comprises a substance for sampling from the surface of the skin. 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 level of a self-integration barrier factor (BANF-1); 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 the self-integration obstacle factor (BANF-1) 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 product is characterized in that the substance for detecting the content of the self-integration barrier factor (BANF-1) also comprises a substance for sampling from the surface of the skin. 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 the self-integration obstacle factor (BANF-1) in the skin sample of the obtained subject;
3) comparing the content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person of the age group, and judging the skin aging degree of the subject according to the comparison result.
Further comprises 4) comparing the content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the standard curve of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person of each age group, judging the physiological age of the 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 level of a self-integration impairment factor (BANF-1) in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store data of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person in accordance with the age group of the subject or the data storage module is configured to store standard curves of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person in each age group;
(3) a data comparison module: the data comparison module is configured to compare the data of the content of the self-integration obstacle factor (BANF-1) in the skin sample of the subject received by the data receiving module with the data of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aged person, 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 data of the content of the self-integration obstacle factor (BANF-1) in the skin sample of the subject received by the data receiving module with the standard curve of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aged person 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 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 a self-integration barrier factor (BANF-1) in a skin sample of a subject, wherein the instrument can be specifically an orbitrap 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 the self-integration obstacle factor (BANF-1) in the skin sample of the obtained subject;
3) comparing the content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the content of the self-integration obstacle factor (BANF-1) 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 content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the standard curve of the content of the self-integration obstacle factor (BANF-1) in normal human skin of each age group, 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 younger than the actual age when the content of the self-integration obstacle factor (BANF-1) in the skin of the subject is higher than that of the self-integration obstacle factor (BANF-1) in the skin of a normal person; the level of the self-integration disorder factor (BANF-1) in the skin of the subject is lower than the level of the self-integration disorder factor (BANF-1) 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.
The invention also provides a method for non-invasively obtaining the self-integration barrier factor in the skin, 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.
The invention further provides a method for determining the relative content of the self-integration barrier factor in the 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 content of the self-integration obstacle factor (BANF-1) in the skin of each age is detected, and the expression of the self-integration obstacle factor (BANF-1) is found to be reduced with the increase of the age, 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 (DFVAEPMGEKPVGSLAGIGEVLGK) of the detected self-integration barrier factor (BANF-1).
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 an application of a self-integration obstacle factor (BANF-1) in auxiliary judgment of aging degree.
The invention provides an application of a substance for detecting the content of a self-integration obstacle factor (BANF-1) in preparing a product; the product has the function of assisting in judging the aging degree.
The invention provides an application of a substance for detecting the content of a self-integration obstacle factor (BANF-1) in preparing a product; the product has the function of assisting in judging the skin aging degree.
The substance for detecting the content of the self-integration obstacle factor (BANF-1) 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 content of the self-integration barrier factor (BANF-1) also comprises a substance for sampling from the surface of the skin.
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 level of a self-integration barrier factor (BANF-1); 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 the self-integration obstacle factor (BANF-1) 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 product is characterized in that the substance for detecting the content of the self-integration barrier factor (BANF-1) also comprises a substance for sampling from the surface of the skin.
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 the self-integration obstacle factor (BANF-1) in the skin sample of the obtained subject;
3) comparing the content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person of the age group, and assisting in judging the skin aging degree of the subject according to the comparison result.
Further comprises 4) comparing the content of the self-integration obstacle factor (BANF-1) measured in the step 2) with the standard curve of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person of each age group, judging the physiological age of the 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 level of a self-integration impairment factor (BANF-1) in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store data of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person in accordance with the age group of the subject or the data storage module is configured to store standard curves of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aging person in each age group;
(3) a data comparison module: the data comparison module is configured to compare the data of the content of the self-integration obstacle factor (BANF-1) in the skin sample of the subject received by the data receiving module with the data of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aged person, 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 data of the content of the self-integration obstacle factor (BANF-1) in the skin sample of the subject received by the data receiving module with the standard curve of the content of the self-integration obstacle factor (BANF-1) in the skin of the normally aged person 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 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 a self-integration barrier factor (BANF-1) in a skin sample of a subject, wherein the instrument can be specifically an orbitrap 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 (0.5x0.5 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) Following centrifugation at 25,000g at 4 ℃ for 15 minutes, the supernatant was recovered and DTT treated with 10mM for 1 hour in a 56 ℃ water bath; (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 16 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 (DFVAEPMGEKPVGSLAGIGEVLGK) of the detected self-integration barrier factor (BANF-1).
The relative levels of the self-integration disorder factor (BANF-1) in the skin samples of the subjects were as follows:
| numbering | Age (age) | Relative content of self-integration barrier factor |
| 1 | 24y | 13.91316161 |
| 2 | 25y | 12.92449999 |
| 3 | 26y | 12.82332349 |
| 4 | 33y | 12.15286442 |
| 5 | 55y | 10.23410373 |
| 6 | 60y | 9.105874097 |
| 7 | 65y | 8.429397179 |
| 8 | 72y | 7.738960222 |
As can be seen from the data in the above table, the relative level of the factor for dysautointegration (BANF-1) in the skin samples of the subjects decreases with age.
In practical application, firstly, the skin of normal people of all ages with statistical significance is collected as samples, the relative content of the self-integration obstacle factor (BANF-1) in each skin sample is respectively measured, a standard curve of the relative content of the age-self-integration obstacle factor (BANF-1) is drawn, then the relative content of the self-integration obstacle factor (BANF-1) in the skin sample of a subject is measured, and the relative content is compared with the standard curve to judge the skin aging degree of the subject. Or to a group of people of a certain age. For example, to serve a 45-year-old population in a city, a statistical sample of the skin of a 45-year-old normal person living in the city is first collected, the relative content of the self-integration disorder factor (BANF-1) 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 the self-integration disorder factor (BANF-1) in the skin is measured by the same method as the threshold value, and when the content of the self-integration disorder factor (BANF-1) is lower than the threshold value, the physiological age of the skin of the subject is older than the actual age; and when the content of the self-integration obstacle factor (BANF-1) in the skin of the subject is higher than a threshold value, judging that the physiological age of the skin of the subject is younger than the actual age.
As to how to measure the amount of the self-integration disorder factor (BANF-1) in the skin, any method capable of determining the absolute and relative amounts of proteins, 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 skin, the content of the self-integration obstacle factor (BANF-1) can also be used as an index for assisting in judging the overall aging degree of the human body.
Gene:BANF1
Protein, Barrier-to-autointegration factor, the amino acid sequence of which is shown in SEQ ID NO.1, and the details are as follows:
MTTSQKHRDFVAEPMGEKPVGSLAGIGEVLGKKLEERGFDKAYVVLGQFLVLKKDEDLFREWLKDTCGANAKQSRDCFGCLREWCDAFL。
SEQUENCE LISTING
<110> Yangsen
<120> BAF protein as a skin rejuvenation protein marker and a noninvasive extraction method thereof
<130> GNCAQ211029
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 89
<212> PRT
<213> Homo sapiens
<400> 1
Met Thr Thr Ser Gln Lys His Arg Asp Phe Val Ala Glu Pro Met Gly
1 5 10 15
Glu Lys Pro Val Gly Ser Leu Ala Gly Ile Gly Glu Val Leu Gly Lys
20 25 30
Lys Leu Glu Glu Arg Gly Phe Asp Lys Ala Tyr Val Val Leu Gly Gln
35 40 45
Phe Leu Val Leu Lys Lys Asp Glu Asp Leu Phe Arg Glu Trp Leu Lys
50 55 60
Asp Thr Cys Gly Ala Asn Ala Lys Gln Ser Arg Asp Cys Phe Gly Cys
65 70 75 80
Leu Arg Glu Trp Cys Asp Ala Phe Leu
85
Claims (10)
1. The application of a substance for detecting the content of the self-integration barrier factor in the preparation of products; the product has the function of assisting in judging the aging degree.
2. The application of a substance for detecting the content of the self-integration barrier factor 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 a self-integration barrier factor; 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 the self-integration obstacle factor is a mass spectrometric identification reagent, an antibody or an antigen binding fragment thereof; and/or the substance for detecting the content of the self-integration barrier factor is an orbitrap 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 the self-integration barrier factor in the skin sample of the obtained subject;
3) comparing the self-integration obstacle factor content measured in the step 2) with the self-integration obstacle factor 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 self-integration obstacle factor content measured in the step 2) with a standard curve of the self-integration obstacle factor content in the skin of the normally aged person of each age, 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 self-integration barrier factors in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store data of the content of the self-integration barrier factor in normal human skin consistent with the age of the subject or the data storage module is configured to store standard curves of the content of the self-integration barrier factor in normal human skin of each age;
(3) a data comparison module: the data comparison module is configured to compare the data of the content of the self-integration barrier factor in the skin sample of the subject received by the data receiving module with the data of the content of the self-integration barrier factor in the skin of the normal person, 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 data of the content of the self-integration barrier factor in the skin sample of the subject received by the data receiving module with the standard curve of the content of the self-integration barrier factor 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 the self-integration barrier factor in the skin sample of the subject; specifically, the substance for detecting the content of the self-integration obstacle factor 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 the self-integration barrier factor in the skin sample of the subject is an orbitrap 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 the self-integration barrier factor in the skin sample of the obtained subject;
3) comparing the self-integration obstacle factor content measured in the step 2) with the self-integration obstacle factor 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 content of the self-integration obstacle factors measured in the step 2) with the standard curve of the content of the self-integration obstacle factors in normal human skin of each age group, 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 older than the actual age, wherein the level of the self-integration barrier factor in the skin of the subject is lower than the level of the self-integration barrier factor in the skin of the normal human; the physiological age of the skin of the subject is judged to be younger than the actual age, in which the content of the self-integration barrier factor in the skin of the subject is higher than that in the skin of a normal person.
9. A method of non-invasively obtaining a self-integration barrier factor in 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 the self-integration barrier factor 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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| CN202110419792.6A Pending CN113189350A (en) | 2021-03-19 | 2021-04-19 | HBB protein as skin rejuvenation protein marker and noninvasive extraction method thereof |
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