CN114231506A - Skin rejuvenation protein marker LX15B protein and noninvasive extraction method thereof - Google Patents
Skin rejuvenation protein marker LX15B protein and noninvasive extraction method thereof Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11033—Arachidonate 15-lipoxygenase (1.13.11.33)
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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
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
- G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
Abstract
The invention discloses a skin rejuvenation protein marker LX15B protein and a noninvasive extraction method thereof. The method aims to find out intrinsic factors of skin rejuvenation from the root, intervene skin aging in advance before appearance of external manifestations of skin aging, keep the skin rejuvenated, and accurately judge the skin rejuvenation degree of the skin and judge whether the physiological age of the skin rejuvenation is consistent with the actual age, so that references and directions are provided for beauty treatment or medical beauty treatment.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a skin rejuvenation protein marker LX15B protein and a noninvasive extraction method thereof.
Background
Criteria for skin rejuvenation: the sebum secretion is moderate, the skin is neither dry nor oily, the skin color is ruddy and fine, the skin is elastic, the thickness is moderate, pores are small, the skin is insensitive to external stimulation, and the pH value is 5-5.6.
With the improvement of living standard, people pay more and more attention to skin care, but usually only pay attention to the external manifestations of skin aging, such as wrinkle, color spot, pore thickness degree and other information, and judge the youth degree of skin from the information.
LX15B is a structurally related member of the lipoxygenase family of non-heme iron dioxygenases, involved in the production of fatty acid hydroperoxides, converting Arachidonic Acid (AA) to 15S-hydroperoxydicarbotetraenoic acid/(15S) -HPETE. It also acts on linoleic acid to produce 13-hydroxyoctadecadienoic acid/13-HPODE. AA plays an important role in the development of epidermal diseases and can synthesize the prostaglandin family through COX-1 and COX-2. AA also synthesizes the leukotriene family by 5-lipoxygenase. Many skin diseases are associated with prostaglandins or related compounds. PGD2 and PDE2 are associated with inflammation and can promote telangiectasia and erythema of the skin. B4(LTB4) is also a well-known inflammatory factor that can promote methicillin-resistant staphylococcus aureus (MRSA) infection via its receptor, B leukotriene receptor 1(BLT 1). In addition, inhibition of the 5-lipoxygenase (5-LO) products of leukotriene B4(LTB4) and cysteinyl leukotrienes (cysLTs) modulates inflammatory responses and improves skin wound healing. As a member of the lipoxygenase family, LX15B plays an important role in AA metabolism, reduces excessive accumulation of AA in the epidermis, greatly reduces inflammatory responses, and is critical for maintaining skin barrier function. Meanwhile, LX15B can produce linoleic acid and 13-hydroxyoctadecadienoic acid/13-HPODE, which can be used to regulate the differentiation of skin cells and eventually form a proper water barrier, thus maintaining normal skin barrier function.
At present, LX15B protein (B type arachidonic acid 15-lipoxygenase) is not used as a precedent for assisting in judging the youthfulness of skin.
Disclosure of Invention
A method for non-invasively extracting LX15B protein from skin, comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
Preferably, the method for determining the relative content of the LX15B protein 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; separation by ULX15B protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
Preferably, the substance for detecting the content of the LX15B protein is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the content of the LX15B protein is an orbital trap high-resolution mass spectrometer.
Preferably, the P value of the LX15B protein is 0.016722493.
Preferably, the method for judging the aging degree and the skin aging degree by the LX15B protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of LX15B protein in the obtained skin sample of the subject;
3) comparing the LX15B protein content measured in the step 2) with the LX15B protein 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;
or 4) comparing the LX15B protein content measured in the step 2) with the standard curve of the LX15B protein 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.
Preferably, the system for assisting in determining the degree of aging comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive LX15B protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store LX15B protein content data in normal human skin consistent with the age bracket of the subject;
(3) a data comparison module: the data comparison module is configured to compare the LX15B protein content data in the skin sample of the subject received by the data receiving module with the LX15B protein content data in the normal human skin, which is consistent with the age group of the subject, stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
The method for detecting the LX15B protein in the skin to assist in judging the youth degree of the skin is simple, accurate in result and high in efficiency. The method is characterized in that the intrinsic factors causing skin aging are found out radically, the skin aging is intervened in advance before the appearance of the external appearance of the skin aging, in addition, the youth degree of the skin can be judged correctly, whether the physiological age of the aging is consistent with the actual age can be judged, and a reference and a direction are provided for beauty treatment or medical beauty treatment.
Drawings
FIG. 1 is a mass spectrum of a characteristic peptide fragment (GFLNQESSGIPSSLETR) of the detected LX15B protein.
Detailed Description
The present invention is further described below by way of specific examples, but the present invention is not limited to only the following examples. Variations, combinations, or substitutions of the invention, which are within the scope of the invention or the spirit, scope of the invention, will be apparent to those of skill in the art and are within the scope of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
A method for non-invasively extracting LX15B protein from skin, comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
The method for measuring the relative content of the LX15B protein 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; separation by ULX15B protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
The substance for detecting the content of the LX15B protein is a mass spectrometric identification reagent, an antibody or an antigen binding fragment thereof; the substance for detecting the content of the LX15B protein is an orbital trap high-resolution mass spectrometer.
The P value of the LX15B protein is 0.016722493.
The method for judging the aging degree and the skin aging degree by the LX15B protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of LX15B protein in the obtained skin sample of the subject;
3) comparing the LX15B protein content measured in the step 2) with the LX15B protein 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;
or 4) comparing the LX15B protein content measured in the step 2) with the standard curve of the LX15B protein 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.
Preferably, the system for assisting in determining the degree of aging comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive LX15B protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store LX15B protein content data in normal human skin consistent with the age bracket of the subject;
(3) a data comparison module: the data comparison module is configured to compare the LX15B protein content data in the skin sample of the subject received by the data receiving module with the LX15B protein content data in the normal human skin, which is consistent with the age group of the subject, stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
FIG. 1 is a mass spectrum of a characteristic peptide fragment (GFLNQESSGIPSSLETR) of the detected LX15B protein.
Randomly sampling 7 women and 6 men of normal healthy Chinese as subjects, wherein the data of the relative content of LX15B protein in skin samples are as follows:
| group A young group (number) | Age (age) | Relative content of LX15B protein |
| 1 | 20y (Man) | 12.58962142 |
| 2 | 24y (woman) | 14.27513498 |
| 3 | 25y (Man) | 11.66451953 |
| 4 | 26y (woman) | 12.96768364 |
| 5 | 27y (woman) | 12.72706294 |
| 6 | 31y (Man) | 12.75902245 |
| 7 | 33y (woman) | 10.65818093 |
| Group B youth group (number) | Age (age) | Relative content of LX15B protein |
| 1 | 55y (Man) | 9.934089093 |
| 2 | 57y (Man) | 11.00602432 |
| 3 | 60y (woman) | 10.95854664 |
| 4 | 63y (woman) | 10.15684274 |
| 5 | 65y (Man) | 10.61058454 |
| 6 | 72y (woman) | 11.31355062 |
As can be seen from the data in the above table, the relative amount of LX15B protein in the skin samples of the subjects decreased with age.
In practical application, firstly, the skin of each statistically significant normal person of each age is collected as a sample, the relative content of the LX15B protein in each skin sample is respectively measured, for example, to serve a group of people of 40 years old in a certain city, then firstly, the skin sample of the statistically significant normal person of 40 years old living in the city is collected, the relative content of the LX15B protein in each skin sample is measured, 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 LX15B protein in the skin is measured by the same method for obtaining the threshold value, and when the content of LX15B protein is higher than the threshold value, the physiological age of the skin of the subject is younger than the actual age; when the LX15B protein content in the skin of the subject is lower than the value, the physiological age of the skin of the subject is judged to be older than the actual age.
As to how to measure the content of LX15B protein in skin, any method capable of determining the absolute and relative content of protein, such as antigen-antibody binding method, etc., other than the method of mass spectrometry in this example, is possible and should be protected by the present invention.
Besides skin, the content of LX15B protein can also be used as an index for assisting in judging the overall aging degree of human.
Gene:LX15B
Protein LX15B Protein
MAEFRVRVSTGEAFGAGTWDKVSVSIVGTRGESPPLPLDNLGKEFTAGAEEDFQVTLPEDVGRVLLLRVHKAPPVLPLLGPLAPDAWFCRWFQLTPPRGGHLLFPCYQWLEGAGTLVLQEGTAKVSWADHHPVLQQQRQEELQARQEMYQWKAYNPGWPHCLDEKTVEDLELNIKYSTAKNANFYLQAGSAFAEMKIKGLLDRKGLWRSLNEMKRIFNFRRTPAAEHAFEHWQEDAFFASQFLNGLNPVLIRRCHYLPKNFPVTDAMVASVLGPGTSLQAELEKGSLFLVDHGILSGIQTNVINGKPQFSAAPMTLLYQSPGCGPLLPLAIQLSQTPGPNSPIFLPTDDKWDWLLAKTWVRNAEFSFHEALTHLLHSHLLPEVFTLATLRQLPHCHPLFKLLIPHTRYTLHINTLARELLIVPGQVVDRSTGIGIEGFSELIQRNMKQLNYSLLCLPEDIRTRGVEDIPGYYYRDDGMQIWGAVERFVSEIIGIYYPSDESVQDDRELQAWVREIFSKGFLNQESSGIPSSLETREALVQYVTMVIFTCSAKHAAVSAGQFDSCAWMPNLPPSMQLPPPTSKGLATCEGFIATLPPVNATCDVILALWLLSKEPGDQRPLGTYPDEHFTEEAPRRSIATFQSRLAQISRGIQERNQGLVLPYTYLDPPLIENSVSI。
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for non-invasively extracting LX15B protein from skin, comprising the steps of:
(1) sampling of skin samples of the epidermis of a subject: sticking the 3M medical adhesive patch to the curved side part of the forearm, and slightly removing the 3M adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) obtaining of a dried peptide fragment sample: 1) cutting the adhesive tape-shaped skin sample into small pieces, depositing on a glass plate, and transferring to a centrifuge tube;
2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) centrifuging at 25,000g centrifugal force at 4 deg.C for 15 minutes, recovering the supernatant and treating DTT with 10mM for 1 hour in a water bath at 56 deg.C;
4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.
2. The method of claim 1, wherein: the method for measuring the relative content of the LX15B protein 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; separation by ULX15B protein LC; the sample was first enriched and desalted on a trap column, then connected in series with a self-contained C18 column, at a flow rate of 500nl/min, by the following effective gradient:
separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;
DDA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5;
DIA mass spectrometric detection
The peptide segment separated by the liquid phase is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-active HF mode for detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.
3. The method for detecting LX15B protein according to claim 2, wherein: the substance for detecting the content of the LX15B protein is a mass spectrometric identification reagent, an antibody or an antigen binding fragment thereof; the substance for detecting the content of the LX15B protein is an orbital trap high-resolution mass spectrometer.
4. The method for detecting LX15B protein according to claim 2, wherein: the P value of the LX15B protein is 0.016722493.
5. The LX15B protein according to claim 1, wherein: the method for judging the aging degree and the skin aging degree by the LX15B protein comprises the following steps:
1) taking a sample of the epidermal skin of a subject;
2) detecting the content of LX15B protein in the obtained skin sample of the subject;
3) comparing the LX15B protein content measured in the step 2) with the LX15B protein 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;
or 4) comparing the LX15B protein content measured in the step 2) with the standard curve of the LX15B protein 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. The method of claim 5, wherein: the system for assisting in judging the aging degree comprises the following modules:
(1) a data receiving module; the data receiving module is configured to receive LX15B protein content data in a skin sample of a subject;
(2) a data storage module: the data storage module is configured to store LX15B protein content data in normal human skin consistent with the age bracket of the subject;
(3) a data comparison module: the data comparison module is configured to compare the LX15B protein content data in the skin sample of the subject received by the data receiving module with the LX15B protein content data in the normal human skin, which is consistent with the age group of the subject, stored in the data storage module;
(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.
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