CN113030490A - Skin rejuvenation protein marker ABRX2 protein and noninvasive extraction method thereof - Google Patents
Skin rejuvenation protein marker ABRX2 protein and noninvasive extraction method thereof Download PDFInfo
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Abstract
The invention discloses a skin rejuvenation protein marker ABRX2 protein and a non-invasive extraction method thereof. The invention is used for detecting the content of the substance Abraxas 2(ABRAXAS2) of the BRISC complex subunit of the subject in preparing the product; the product has the function of judging or assisting in judging skin aging. The invention is used for detecting the application of the substance of BRISC complex subunit Abraxas2 content in the preparation of products; the product has the function of diagnosing or assisting in diagnosing the skin aging degree. The method judges the skin aging degree by detecting the content of the BRISC complex subunit Abraxas2 of the person to be detected, and is simple, convenient, accurate in result and high in efficiency; it provides basis for skin aging degree of subjects of different ages and sexes, and plays a guiding role in skin management.
Description
Technical Field
The invention relates to a skin rejuvenation protein marker ABRX2 protein and a noninvasive extraction method thereof, belonging to the field of molecular biology.
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.
If the skin is not well maintained or deteriorates with age, dead skin adheres to the surface of the skin without falling off, thereby causing a series of problems and seriously affecting beauty. With the improvement of living standard of people, people pay more and more attention to skin care, if the skin can not be well maintained or declines with age, dead skin can be attached to the surface of the skin and can not fall off, thereby causing a series of problems and seriously affecting beauty. However, the method for judging the degree of skin aging usually focuses only on the external manifestations of skin aging, such as wrinkles, spots, and pore thickness, and thus judges the degree of skin aging, and this method cannot find the intrinsic factors causing skin aging at the root, cannot intervene in skin aging in advance before the external manifestations of skin aging appear, and is also a precondition for beauty treatment or medical beauty treatment to judge the degree of skin aging correctly and judge whether the physiological age of aging coincides with the actual age.
The ABRAXAS2 protein (BRISC complex subunit ABRAXAS2, ABRAXAS2) is a scaffold protein that recruits various peptides to assemble the BRCC36 isopeptide complex (BRISC) Deubiquitinase (DUB) complex. Inhibitors of DUBS have been shown to induce autophagy and thus inhibit cell death. The BRISC enzyme has deubiquitinating activity and consists of four known subunits: MERIT 40; BRE expressed in the brain and reproductive organs; BRCA1/BRCA2 Complex 3 subunit (BRCC 3); ABRAXAS2 is also a newly discovered p53 dependent regulator, and plays an important role in tumor suppression and DNA damage response; consistent with these effects, studies indicate that ABRO1 is involved in telomere replication and genome repair. Hindered chromosomal replication forks are essential for maintaining chromosomal stability. ABRO1 protects the integrity of the blocked replication fork, thereby maintaining genomic stability.
However, there has been no report of judging the degree of skin aging by the ABRX2 protein so far to further study the way of skin rejuvenation.
Disclosure of Invention
The invention aims to provide an ABRX2 protein as a skin rejuvenation protein marker and a non-invasive extraction method thereof.
The invention discloses application of a BRISC complex subunit Abraxas 2(ABRAXAS2) in auxiliary judgment of aging degree.
In the invention, the amino acid sequence of the BRISC complex subunit Abraxas 2(ABRAXAS2) is shown in SEQ ID NO. 1.
The invention provides an application of a substance for detecting the content of BRISC complex subunit Abraxas 2(ABRAXAS2) in preparing products; the product has the function of judging or assisting in judging skin aging.
The invention provides an application of a substance for detecting the content of BRISC complex subunit Abraxas2 in a subject in preparing a product; the product has the function of diagnosing or assisting in diagnosing the skin aging degree.
In the above application, the product is a kit or a chip.
The product comprises a substance for detecting the content of the BRISC complex subunit Abraxas 2; the function of the product is as follows (a) or (b):
(a) judging or assisting in judging skin aging;
(b) diagnosing or aiding in the diagnosis of the degree of skin aging.
In the above product, the substance for detecting the content of the complex subunit Abraxas2 of BRISC in the subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof.
In the above product, the apparatus for detecting the content of the BRISC complex subunit Abraxas2 in the subject is an orbitrap high resolution mass spectrometer.
In the above product, the product further comprises a carrier describing the following detection method:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of the BRISC complex subunit Abraxas2 in the skin sample of the obtained subject;
3) comparing the content of the BRISC complex subunit Abraxas2 measured in the step 2) with the content of the BRISC complex subunit Abraxas2 in normal human skin of the same sex of the age group, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the content of the BRISC complex subunit Abraxas2 measured in the step 2) with a standard curve of the content of the BRISC complex subunit Abraxas2 in normal human skin of the same sex at each age, and judging the aging degree of the skin or the physiological age of the skin of the subject according to the comparison result.
The invention further provides a data processing device system for assisting in judging the aging degree, which comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive BRISC complex subunit Abraxas2 content data 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 BRISC complex subunit Abraxas2 in the skin of a normal person consistent with the age and the sex of the subject or the data storage module is configured to store a standard curve of the content of the BRISC complex subunit Abraxas2 in the skin of a normal person with different sex;
(3) a data comparison module: the data comparison module is configured to compare the BRISC complex subunit Abraxas2 content data in the skin sample of the subject received by the data receiving module with the BRISC complex subunit Abraxas2 content data in normal human skin, which is consistent with the age and sex of the subject, stored in the data storage module; or the data comparison module is configured to compare the BRISC complex subunit Abraxas2 content data in the skin sample of the subject received by the data receiving module with a BRISC complex subunit Abraxas2 content standard curve in normal human skin of different sex 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.
In the data processing device system, the system for assisting in determining the aging degree further comprises a substance for detecting the content of the BRISC complex subunit Abraxas2 in the skin sample of the subject;
the substance for detecting the content of the BRISC complex subunit Abraxas2 in the subject can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof.
In the data processing device system, the system for assisting in determining the aging degree further comprises an instrument for detecting the content of the BRISC complex subunit Abraxas2 in the skin sample of the subject;
the instrument for detecting the content of the BRISC complex subunit Abraxas2 in the skin sample of the subject can be an orbital trap high-resolution mass spectrometer.
The invention provides a method for extracting peptide fragments in BRISC complex subunit Abraxas2 in a skin sample of a subject, which comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) the taped skin samples were cut into small pieces, applied with a sterile razor blade and deposited on glass plates before being transferred to a 1.5 ml centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, and then placing 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 under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;
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 BRISC complex subunit Abraxas2 in a skin sample of a subject, which comprises the following steps:
(1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) the taped skin samples were cut into small pieces, applied with a sterile razor blade and deposited on glass plates before being transferred to a 1.5 ml centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, and then placing 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 under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;
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
a) 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 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 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
b) DDA and/or DIA mass spectrometric detection
Detecting DDA mass spectrum:
ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, CA) to perform 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: 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 is set to 30s, the AGC is set to: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection:
ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DIA (data-independent acquisition) mode detection; the parameters are set as follows: 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 invention has the following advantages:
the skin aging degree is judged by comparing the BRISC complex subunit Abraxas2 content of a person to be detected with a standard curve, the method is simpler and more convenient, the result is accurate, and the efficiency is high; it provides basis for skin aging degree of subjects of different ages and sexes, and plays a guiding role in skin management.
Drawings
FIG. 1 is a mass spectrum of a characteristic peptide fragment (AIYQVYNALQEK) of the BRISC complex subunit Abraxas 2(ABRAXAS2) of the present invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
The white-collar people with relatively close living environment and healthy body are taken as samples, and the epidermal skin samples are sampled by a noninvasive method (specifically 3M adhesive skin is used for completing sampling).
The method for measuring the protein content in the epidermal skin sample of the subject comprises the following steps:
(1) high performance liquid phase
Sampling of skin samples of the epidermis of a subject: sticking 3M glue (3M Health Care 3M Brookings from 3M medical glue paste manufacturers) to the curved side part of the forearm to eliminate light aging factors; after 1 minute, the 3M adhesive patch was gently removed to ensure that the skin of the subject was not damaged, and a taped skin sample was obtained.
Obtaining of a dried peptide fragment sample: 1) tape-like skin samples were cut into small pieces (0.5 x 0.5 cm), applied with a sterile razor blade and deposited on glass plates before transfer 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 under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;
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 chromatography column and drying under vacuum; thus obtaining the dried peptide fragment sample.
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; 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 peptide fragments after liquid phase separation were ionized by 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 are ionized by a nanoESI source and then enter 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.
The mass spectrum of the characteristic peptide fragment (AIYQVYNALQEK) of the complex subunit of bresc Abraxas 2(Abraxas2) as determined by the above method is shown in fig. 1.
The BRISC complex subunit Abraxas 2(Abraxas2) was measured in skin samples from male and female subjects, respectively, as described above, and the relative content data based on mass spectra are shown in tables 1 and 2 below:
TABLE 1 relative mass-spectrometric-based content of ABRAXAS2 in skin samples of male subjects
Age (year of old) | Relative content of ABRAXAS2 based on mass spectrum |
20 | 13.600359666816 |
25 | 13.5595567080337 |
55 | 13.449518921758 |
57 | 13.2444414634172 |
65 | 12.5867348597154 |
TABLE 2 relative mass-spectrometric-based content of ABRAXAS2 in skin samples of female subjects
Age (year of old) | Relative content of ABRAXAS2 based on mass spectrum |
24 | 13.9501694402891 |
26 | 13.839600372192 |
60 | 12.9269874018133 |
63 | 12.8736624028962 |
72 | 12.7774123033345 |
As can be seen from the data in table 1 above, the relative content of the BRISC complex subunit Abraxas 2(Abraxas2) in the skin samples of male subjects decreased with age based on mass spectrometry. As can be seen from the data in table 2 above, the relative content of the BRISC complex subunit Abraxas 2(Abraxas2) in skin samples of female subjects decreased with age based on mass spectrometry.
In practical application, the method for assisting in judging the skin aging degree of a subject according to the relative content of the BRISC complex subunit Abraxas 2(ABRAXAS2) based on mass spectrum is as follows:
(1) firstly, collecting a large number of skin samples of normal people with statistical significance in each age stage of male or female, and respectively determining the relative content of ABRAXAS2 in each skin sample based on mass spectrum;
(2) determining the relative mass-spectrometric-based content of ABRAXAS2 in a skin sample from the subject; then carrying out (3) or (4);
(3) comparing the data of the subject in the step (2) with the ABRAXAS2 in normal human skin of the same sex in the age group based on the content value of the mass spectrum, and judging the relative skin aging degree of the subject according to the comparison result;
(4) and (3) drawing a standard curve of male or female age-ABRAXAS 2 based on the relative content of mass spectra based on the data in the step (1), measuring the relative content of ABRAXAS2 in the skin sample of the subject based on the mass spectra, and comparing the relative content with the standard curve to judge the skin aging degree or skin age of the subject.
In the present invention, the above-mentioned BRISC complex subunit Abraxas2 is specifically shown as follows:
Gene:ABRAXAS2
Protein:BRISC complex subunit Abraxas 2
the amino acid sequence is shown as SEQ ID NO.1, and the amino acid sequence is as follows:
MAASISGYTFSAVCFHSANSNADHEGFLLGEVRQEETFSISDSQISNTEFLQVIEI HNHQ
PCSKLFSFYDYASKVNEESLDRILKDRRKKVIGWYRFRRNTQQQMSYREQVLH KQLTRIL
GVPDLVFLLFSFISTANNSTHALEYVLFRPNRRYNQRISLAIPNLGNTSQQEYKV SSVPN
TSQSYAKVIKEHGTDFFDKDGVMKDIRAIYQVYNALQEKVQAVCADVEKSER VVESCQAE
VNKLRRQITQRKNEKEQERRLQQAVLSRQMPSESLDPAFSPRMPSSGFAAEGRS TLGDAE
ASDPPPPYSDFHPNNQESTLSHSRMERSVFMPRPQAVGSSNYASTSAGLKYPGS GADLPP
PQRAAGDSGEDSDDSDYENLIDPTEPSNSEYSHSKDSRPMAHPDEDPRNTQTS QI 。
sequence listing
<110> Yangsen
<120> skin rejuvenation protein marker-ABRX 2 protein and noninvasive extraction method thereof
<130>GNCLW211024
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<170>PatentIn version 3.5
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Ser Ala Asn Ser Asn Ala Asp His Glu Gly Phe Leu Leu Gly Glu Val
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Arg Gln Glu Glu Thr Phe Ser Ile Ser Asp Ser Gln Ile Ser Asn Thr
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Glu Phe Leu Gln Val Ile Glu Ile His Asn His Gln Pro Cys Ser Lys
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Leu Phe Ser Phe Tyr Asp Tyr Ala Ser Lys Val Asn Glu Glu Ser Leu
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Phe Arg Arg Asn Thr Gln Gln Gln Met Ser Tyr Arg Glu Gln Val Leu
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His Lys Gln Leu Thr Arg Ile Leu Gly Val Pro Asp Leu Val Phe Leu
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Leu Phe Ser Phe Ile Ser Thr Ala Asn Asn Ser Thr His Ala Leu Glu
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Tyr Val Leu Phe Arg Pro Asn Arg Arg Tyr Asn Gln Arg Ile Ser Leu
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Ala Ile Pro Asn Leu Gly Asn Thr Ser Gln Gln Glu Tyr Lys Val Ser
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Ser Val Pro Asn Thr Ser Gln Ser Tyr Ala Lys Val Ile Lys Glu His
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Ile Tyr Gln Val Tyr Asn Ala Leu Gln Glu Lys Val Gln Ala Val Cys
210 215 220
Ala Asp Val Glu Lys Ser Glu Arg Val Val Glu Ser Cys Gln Ala Glu
225 230 235 240
Val Asn Lys Leu Arg Arg Gln Ile Thr Gln Arg Lys Asn Glu Lys Glu
245 250 255
Gln Glu Arg Arg Leu Gln Gln Ala Val Leu Ser Arg Gln Met Pro Ser
260 265 270
Glu Ser Leu Asp Pro Ala Phe Ser Pro Arg Met Pro Ser Ser Gly Phe
275 280 285
Ala Ala Glu Gly Arg Ser Thr Leu Gly Asp Ala Glu Ala Ser Asp Pro
290 295 300
Pro Pro Pro Tyr Ser Asp Phe His Pro Asn Asn Gln Glu Ser Thr Leu
305 310 315 320
Ser His Ser Arg Met Glu Arg Ser Val Phe Met Pro Arg Pro Gln Ala
325 330 335
Val Gly Ser Ser Asn Tyr Ala Ser Thr Ser Ala Gly Leu Lys Tyr Pro
340 345 350
Gly Ser Gly Ala Asp Leu Pro Pro Pro Gln Arg Ala Ala Gly Asp Ser
355 360 365
Gly Glu Asp Ser Asp Asp Ser Asp Tyr Glu Asn Leu Ile Asp Pro Thr
370 375 380
Glu Pro Ser Asn Ser Glu Tyr Ser His Ser Lys Asp Ser Arg Pro Met
385 390 395 400
Ala His Pro Asp Glu Asp Pro Arg Asn Thr Gln Thr Ser Gln Ile
405 410 415
Claims (10)
1. The application of a substance for detecting the content of the BRISC complex subunit Abraxas2 in a subject in preparing a product; the product has the function of judging or assisting in judging skin aging.
2. The application of a substance for detecting the content of the BRISC complex subunit Abraxas2 in a subject in preparing a product; the product has the function of diagnosing or assisting in diagnosing the skin aging degree.
3. A product comprising a substance for detecting the content of bresc complex subunit Abraxas 2; the function of the product is as follows (a) or (b):
(a) judging or assisting in judging skin aging;
(b) diagnosing or aiding in the diagnosis of the degree of skin aging.
4. The product of claim 3, wherein: the product is a kit or a chip; and/or
The substance for detecting the content of the BRISC complex subunit Abraxas2 in the subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; and/or
The instrument for detecting the content of the BRISC complex subunit Abraxas2 in the subject is an orbital trap high-resolution mass spectrometer.
5. The product according to claim 3 or 4, characterized in that: 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 BRISC complex subunit Abraxas2 in the skin sample of the obtained subject;
3) comparing the content of the BRISC complex subunit Abraxas2 measured in the step 2) with the content of the BRISC complex subunit Abraxas2 in normal human skin of the same sex of the age group, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the content of the BRISC complex subunit Abraxas2 measured in the step 2) with a standard curve of the content of the BRISC complex subunit Abraxas2 in normal human skin of the same sex at each age, and judging the skin aging degree or the physiological age of the skin of the subject according to the comparison result.
6. A data processing device 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 BRISC complex subunit Abraxas2 content data 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 BRISC complex subunit Abraxas2 in the skin of a normal person consistent with the age and the sex of the subject or the data storage module is configured to store a standard curve of the content of the BRISC complex subunit Abraxas2 in the skin of a normal person with different sex;
(3) a data comparison module: the data comparison module is configured to compare the BRISC complex subunit Abraxas2 content data in the skin sample of the subject received by the data receiving module with the BRISC complex subunit Abraxas2 content data in normal human skin, which is consistent with the age and sex of the subject, stored in the data storage module; or the data comparison module is configured to compare the BRISC complex subunit Abraxas2 content data in the skin sample of the subject received by the data receiving module with a BRISC complex subunit Abraxas2 content standard curve in normal human skin of different sex 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 data processing apparatus system of claim 6, wherein: the system for assisting in judging the aging degree also comprises a substance for detecting the content of the BRISC complex subunit Abraxas2 in a skin sample of a subject;
the system for assisting in judging the aging degree further comprises an instrument for detecting the content of the BRISC complex subunit Abraxas2 in the skin sample of the subject;
the substance for detecting the content of the BRISC complex subunit Abraxas2 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 BRISC complex subunit Abraxas2 in the skin sample of the subject is specifically an orbitrap high-resolution mass spectrometer.
8. The method for assisting in judging the degree of skin aging comprises the following steps:
1) sticking the adhesive on the skin surface of the forearm curved side part, and tearing off to obtain a skin sample of the epidermis of the subject;
2) detecting the hepatic globin content in the skin sample of the obtained subject;
3) comparing the content of the BRISC complex subunit Abraxas2 measured in the step 2) with the content of the BRISC complex subunit Abraxas2 in the skin of a normal person with the same age and sex of the subject, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the BRISC complex subunit Abraxas2 content measured in the step 2) with a standard curve of the BRISC complex subunit Abraxas2 content in normal human skin with the same age and sex, 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, wherein the content of the BRISC complex subunit Abraxas2 in the skin of the subject is lower than the content of the BRISC complex subunit Abraxas2 in normal human skin; the content of the BRISC complex subunit Abraxas2 in the skin of the subject is higher than that of the BRISC complex subunit Abraxas2 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 for extracting peptide fragments in BRISC complex subunit Abraxas2 in a skin sample of epidermis of a subject comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) the taped skin samples were cut into small pieces, applied with a sterile razor blade and deposited on glass plates before being transferred to a 1.5 ml centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, and then placing 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 under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;
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. A method for determining the mass spectrum-based relative content of the BRISC complex subunit Abraxas2 in an epidermal skin sample of a subject, comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) the taped skin samples were cut into small pieces, applied with a sterile razor blade and deposited on glass plates before being transferred to a 1.5 ml centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, and then placing 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 under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;
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
a) 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 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 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
b) DDA and/or DIA mass spectrometric detection
Detecting DDA mass spectrum:
ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, CA) to perform 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: 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 is set to 30s, the AGC is set to: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection:
ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DIA (data-independent acquisition) mode detection; the parameters are set as follows: 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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