CN116144621A - Skin rejuvenation protein marker ACLY protein and noninvasive extraction method thereof - Google Patents

Skin rejuvenation protein marker ACLY protein and noninvasive extraction method thereof Download PDF

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CN116144621A
CN116144621A CN202110469444.XA CN202110469444A CN116144621A CN 116144621 A CN116144621 A CN 116144621A CN 202110469444 A CN202110469444 A CN 202110469444A CN 116144621 A CN116144621 A CN 116144621A
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杨森
张学军
张博
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Abstract

The invention discloses an ACLY protein as a skin rejuvenation protein marker and a non-invasive extraction method thereof. The extraction detection method and application of ACLY protein in auxiliary judgment of skin rejuvenation degree are disclosed, aiming at finding the intrinsic factor of skin rejuvenation from root, intervening skin aging in advance before appearance of skin aging appearance, keeping younger skin, in addition, can correctly judge skin rejuvenation degree of skin and judge whether physiological age of skin rejuvenation accords with actual age, and provide reference and direction for beauty or medical beauty.

Description

Skin rejuvenation protein marker ACLY protein and noninvasive extraction method thereof
Technical Field
The invention relates to the field of molecular biology, in particular to an ACLY protein which is a skin rejuvenation protein marker and a noninvasive extraction method thereof.
Background
Criteria for skin rejuvenation: the skin color is ruddy and fine, has elasticity, moderate thickness, smaller pores, is insensitive to external stimulus and has a PH value of 5-5.6.
With the improvement of living standard, people pay more attention to skin care, but usually only pay attention to the external appearance of skin aging, such as wrinkles, color spots, pore size and other information, and thus the skin youth degree is judged, the method for judging the skin youth degree cannot fundamentally find the reason for keeping the skin youthful, cannot intervene in skin aging in advance before the external appearance of skin aging appears, and in addition, the method for accurately judging the skin youth degree and judging whether the physiological age of aging accords with the actual age is also a precondition of beauty or medical beauty.
ACLY is ATP citrate synthase, the primary enzyme responsible for the synthesis of many tissue cytoplasmic acetyl-coa. In previous studies, ACLY was found to be up-regulated in normal human skin fibroblasts and normal human skin keratinocytes when a series of well known purified antioxidants were applied for 24 hours. Most antioxidants may affect skin lipid synthesis, whereas acetyl-coa is an important component of sebum synthesis and can be used in a number of important biosynthetic pathways, including adipogenesis and cholesterol production. Cholesterol, which is a keratinocyte lipid, is an important component in the attachment and stabilization of the epidermis. It plays an important role in maintaining stability and moisture retention. We speculate that ACLY is primarily involved in the production of fat and cholesterol, which is critical to maintaining stability of skin barrier function and maintaining youthful skin.
No prior art exists for using ACLY protein (ATP citrate lyase) to aid in judging skin youth.
Disclosure of Invention
A method for non-invasively extracting ACLY protein from skin, the method comprising the steps of:
(1) Sampling of a subject epidermis skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample.
Preferably, the method for determining the relative content of ACLY protein in an epidermal skin sample based on mass spectrometry comprises the following steps: (1) sampling of a subject epidermal skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample;
(3) Detection of
Re-dissolving the dried peptide fragment sample with mobile phase A (2% ACN,0.1% FA), centrifuging for 10 min at 20,000g, and collecting supernatant; separation by UACLY protein LC; the sample was first run into a trap column for enrichment and desalting, then serially connected to a self-contained C18 column, at a flow rate of 500nl/min through the following effective gradient:
separating: 0-5min,5% mobile phase B (98% acn,0.1% fa); 5-160min, the mobile phase B is linearly increased from 5% to 35%;160-170min, mobile phase B rises from 35% to 80%;170-175min,80% mobile phase B;176-180min,5% mobile phase B; the nano liter liquid phase separation tail end is directly connected with a mass spectrometer;
DDA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum 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 secondary fragmentation parent ions are as follows: charge 2+ to 7+, parent ion with peak intensity exceeding 10,000 intensity row 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 1E5;
DIA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum is 350-1500 m/z; the resolution is set to 120,000; 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 is set to 30s; the AGC is set to: primary 3E6, secondary 1E5.
Preferably, the substance used to detect ACLY protein content is a mass spectrometry reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the ACLY protein content is an orbitrap high-resolution mass spectrometer.
Preferably, the ACLY protein has a P value of 0.005292344.
Preferably, the method for judging the aging degree and the skin aging degree by using the ACLY protein comprises the following steps:
1) Taking a skin sample of the epidermis of a subject;
2) Detecting the ACLY protein content in the skin sample of the subject;
3) Comparing the ACLY protein content measured in the step 2) with the ACLY protein content value in the skin of the aged person, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the ACLY protein content measured in the step 2) with an ACLY protein content standard curve in the skin of the normal aging personnel 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 judging the aging degree comprises the following modules:
(1) A data receiving module; the data receiving module is configured to receive ACLY protein content data in a skin sample of a subject;
(2) And a data storage module: the data storage module is configured to store ACLY protein content data in normal human skin consistent with a subject's age range;
(3) And a data comparison module: the data comparison module is configured to compare the ACLY protein content data in the skin sample of the subject received by the data receiving module with the ACLY protein content data in the skin of the normal person consistent with the age of the subject stored in the data storage module;
(4) A judging 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, judge whether the skin physiological age of the subject accords with the actual age of the subject, and output the judgment result.
The method for detecting ACLY protein in skin assists in judging skin youth, is simpler and more convenient, and has accurate results and high efficiency. The intrinsic factors causing skin aging are found from the root, the skin aging is intervened in advance before the appearance of the skin aging, in addition, the youth degree of the skin and whether the physiological age of the aging accords with the actual age can be correctly judged, and the reference and the direction are provided for the beauty or medical beauty.
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FIG. 1 is a mass spectrum of a characteristic peptide fragment (QHFPATPLLDYALEVEK) of ACLY protein detected.
Detailed Description
The present invention is further described below by way of specific examples, but the present invention is not limited to the following examples only. Modifications, combinations, or substitutions of the present invention within the scope of the invention or without departing from the spirit and scope of the invention will be apparent to those skilled in the art and are included within the scope of the invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.
A method for non-invasively extracting ACLY protein from skin, the method comprising the steps of:
(1) Sampling of a subject epidermis skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample.
The method for measuring the relative content of ACLY protein in the epidermis skin sample based on mass spectrum comprises the following steps: (1) sampling of a subject epidermal skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample;
(3) Detection of
Re-dissolving the dried peptide fragment sample with mobile phase A (2% ACN,0.1% FA), centrifuging for 10 min at 20,000g, and collecting supernatant; separation by UACLY protein LC; the sample was first run into a trap column for enrichment and desalting, then serially connected to a self-contained C18 column, at a flow rate of 500nl/min through the following effective gradient:
separating: 0-5min,5% mobile phase B (98% acn,0.1% fa); 5-160min, the mobile phase B is linearly increased from 5% to 35%;160-170min, mobile phase B rises from 35% to 80%;170-175min,80% mobile phase B;176-180min,5% mobile phase B; the nano liter liquid phase separation tail end is directly connected with a mass spectrometer;
DDA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum 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 secondary fragmentation parent ions are as follows: charge 2+ to 7+, parent ion with peak intensity exceeding 10,000 intensity row 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 1E5;
DIA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum is 350-1500 m/z; the resolution is set to 120,000; 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 is set to 30s; the AGC is set to: primary 3E6, secondary 1E5.
The substance for detecting the content of ACLY protein is mass spectrum identification reagent, antibody or antigen binding fragment thereof; the substance for detecting the ACLY protein content is an orbitrap high-resolution mass spectrometer.
The P value of the ACLY protein is 0.005292344.
The method for judging the aging degree and the skin aging degree by using the ACLY protein comprises the following steps:
1) Taking a skin sample of the epidermis of a subject;
2) Detecting the ACLY protein content in the skin sample of the subject;
3) Comparing the ACLY protein content measured in the step 2) with the ACLY protein content value in the skin of the aged person, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the ACLY protein content measured in the step 2) with an ACLY protein content standard curve in the skin of the normal aging personnel 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 judging the aging degree comprises the following modules:
(1) A data receiving module; the data receiving module is configured to receive ACLY protein content data in a skin sample of a subject;
(2) And a data storage module: the data storage module is configured to store ACLY protein content data in normal human skin consistent with a subject's age range;
(3) And a data comparison module: the data comparison module is configured to compare the ACLY protein content data in the skin sample of the subject received by the data receiving module with the ACLY protein content data in the skin of the normal person consistent with the age of the subject stored in the data storage module;
(4) A judging 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, judge whether the skin physiological age of the subject accords with the actual age of the subject, and output the judgment result.
FIG. 1 is a mass spectrum of a characteristic peptide fragment (QHFPATPLLDYALEVEK) of ACLY protein detected.
Randomly sampling normal healthy Chinese 7 female and 6 male subjects, wherein the relative content of ACLY protein in skin samples is as follows:
group A young group (number) Age of Relative content of ACLY protein
1 20y (Man) 11.98241316
2 24y (female) 13.0814456
3 25y (Man) 10.36573684
4 26y (female) 11.14189546
5 26y (female) 12.20723523
6 31y (Man) 12.72423826
7 33y (female) 11.3158785
Group B youth group (number) Age of Relative content of ACLY protein
1 52y (Man) 10.54654134
2 57y (Man) 12.78630048
3 60y (female) 9.678019608
4 63y (female) 9.2541871258
5 65y (Man) 8.955421251
6 72y (female) 10.574448716
As can be seen from the data in the table above, the relative levels of ACLY protein in skin samples of subjects decreased with age.
In practical application, firstly, skin of normal people of all ages with statistical significance is collected as a sample, and the relative content of ACLY protein in each skin sample is measured respectively, for example, to serve the crowd of 40 years in a certain city, then firstly, skin sample of normal people of 40 years living in the city with statistical significance is collected, and the relative content of ACLY protein in each skin sample is measured and averaged. The average value is a threshold value for measuring the skin aging degree of a subject, when the subject is evaluated, the content of ACLY protein in the skin is measured by adopting the same method for obtaining the threshold value, and when the content of the ACLY protein is higher than the threshold value, the physiological age of the skin of the subject is lower than the actual age; when the ACLY 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 ACLY protein in the skin, any method capable of determining the absolute and relative content of protein, such as antigen-antibody binding method, etc., other than the method of mass spectrometry in this example, is possible and shall be protected by the present invention.
Besides skin, the content of ACLY protein can also be used as an index for assisting in judging the overall aging degree of a person.
Gene:ACLY
Protein ACLY Protein
MSAKAISEQTGKELLYKFICTTSAIQNRFKYARVTPDTDWARLLQDHPWLLSQNLVVKPDQLIKRRGKLGLVGVNLTLDGVKSWLKP RLGQEATVGKATGFLKNFLIEPFVPHSQAEEFYVCIYATREGDVLFHHEGGVDVGDVDAKAQKLLVGVDEKLNPEDIKKHLLVHAPEDKKE ILASFISGLFNFYEDLYFTYLEINPLVVTKDGVYVLDLAAKVDATADYICKVKWGDIEFPPPFGREAYPEEAYIADLDAKS
GASLKLTLLNPKGRIWTMVAGGGASVVYSDTICDLGGVNELANYGEYSGAPSEQQTYDYAKTILSLMTREKHPDGKILIIGGSIANF TNVAATFKGIVRAIRDYQGPLKEHEVTIFVRRGGPNYQEGLRVMGEVGKTTGIPIHVFGTETHMTAIVGMALGHRPIPNQPPTAAHTANFL LNASGSTSTPAPSRTASFSESRADEVAPAKKAKPAMPQDSVPSPRSLQGKSTTLFSRHTKAIVWGMQTRAVQGMLDFDYVCSRDEPSVAAM VYPFTGDHKQKFYWGHKEILIPVFKNMADAMRKHPEVDVLINFASLRSAYDSTMETMNYAQIRTIAIIAEGIPEALTRKLIKKADQKGVTI IGPATVGGIKPGCFKIGNTGGMLDNILASKLYRPGSVAYVSRSGGMSNELNNIISRTTDGYEGVAIGGDRYPGSTFMDHVLRYQDTPGVKM IVVLGEIGGTEEYKICRGIKEGRLTKPIVCWCIGTCATMFSSEVQFGHAGACANQASETAVAKNQALKEAGVFVPRSFDELGEIIQSVYED LVANGVIVPAQEVPPPTVPMDYSWARELGLIRKPASFMTSICDERGQELIYAGMPITEVFKEEMGIGGVLGLLWFQKLPKYSCQFIEMCLM VTAHGPAVSGAHNTIICARAGKDLVSSLTSGLLTIGDRFGGALDAAAKMFSKAFDSGIIPMEFVNKMKKEGKLIMGIGHRVKSINNPDMRV
QILKDYVRQHFPATPLLDYALEVEKITTSKKPNLILNVDGLIGVAFVDMLRNCGSFTREEADEYIDIGALNGI
FVLGRSMGFIGHYLDQKRLKQGLYRHPWDDISYVLPEHMS。
The above description is illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention, but is to be accorded the full scope of the claims.

Claims (6)

1. A method for non-invasively extracting ACLY protein from skin, the method comprising the steps of:
(1) Sampling of a subject epidermis skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample.
2. The method according to claim 1, characterized in that: the method for measuring the relative content of ACLY protein in the epidermis skin sample based on mass spectrum comprises the following steps: (1) sampling of a subject epidermal skin sample: sticking a 3M medical adhesive patch on the curved side of the forearm, and slightly tearing off the 3M medical adhesive patch after 1 minute to obtain a sticky tape-shaped skin sample;
(2) Obtaining 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,1XCocktail, then placing on ice for 5 minutes, then adding 10mM DTT, and soaking the sample overnight;
3) Centrifuging at 25,000g for 15 min at 4deg.C, recovering supernatant and placing in a water bath at 56 deg.C with 10mM DTT for 1 hr;
4) Then treated with 55mM IAM, incubated for 45 minutes in the dark at room temperature and centrifuged at 25,000g for 15 minutes at 4℃to give the final protein solution supernatant; protein concentration was measured using Bradford method and the extracted proteins were quality controlled by 12% SDS-PAGE; 100. Mu.g of protein was taken per sample, trypsin was added and hydrolyzed at 37℃for 4 hours; then trypsin is added again in the same proportion and enzymolysis is carried out for 8 hours at 37 ℃; desalting the hydrolysate with Strata X chromatographic column, and drying under vacuum to obtain dried peptide sample;
(3) Detection of
Re-dissolving the dried peptide fragment sample with mobile phase A (2% ACN,0.1% FA), centrifuging for 10 min at 20,000g, and collecting supernatant; separation by UACLY protein LC; the sample was first run into a trap column for enrichment and desalting, then serially connected to a self-contained C18 column, at a flow rate of 500nl/min through the following effective gradient:
separating: 0-5min,5% mobile phase B (98% acn,0.1% fa); 5-160min, the mobile phase B is linearly increased from 5% to 35%;160-170min, mobile phase B rises from 35% to 80%;170-175min,80% mobile phase B;176-180min,5% mobile phase B; the nano liter liquid phase separation tail end is directly connected with a mass spectrometer;
DDA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum 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 secondary fragmentation parent ions are as follows: charge 2+ to 7+, parent ion with peak intensity exceeding 10,000 intensity row 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 1E5;
DIA mass spectrometry detection
The peptide segment after liquid phase separation is ionized by a nanoESI source and then is imported to a tandem mass spectrometer Q-exact HF mode for detection; main parameter setting: the ion source voltage is set to be 1.6kV; the scanning range of the primary mass spectrum is 350-1500 m/z; the resolution is set to 120,000; 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 is set to 30s; the AGC is set to: primary 3E6, secondary 1E5.
3. The ACLY protein assay of claim 2, wherein: the substance for detecting the content of ACLY protein is mass spectrum identification reagent, antibody or antigen binding fragment thereof; the substance for detecting the ACLY protein content is an orbitrap high-resolution mass spectrometer.
4. The ACLY protein assay of claim 2, wherein: the P value of the ACLY protein is 0.005292344.
5. The ACLY protein of claim 1, wherein: the method for judging the aging degree and the skin aging degree by using the ACLY protein comprises the following steps:
1) Taking a skin sample of the epidermis of a subject;
2) Detecting the ACLY protein content in the skin sample of the subject;
3) Comparing the ACLY protein content measured in the step 2) with the ACLY protein content value in the skin of the aged person, and judging the skin aging degree of the subject according to the comparison result;
or 4) comparing the ACLY protein content measured in the step 2) with an ACLY protein content standard curve in the skin of the normal aging personnel of each age group, and judging the physiological age of the skin of the subject according to the comparison result.
6. The method according to claim 5, wherein: a system for aiding in determining the degree of aging, comprising:
(1) A data receiving module; the data receiving module is configured to receive ACLY protein content data in a skin sample of a subject;
(2) And a data storage module: the data storage module is configured to store ACLY protein content data in normal human skin consistent with a subject's age range;
(3) And a data comparison module: the data comparison module is configured to compare the ACLY protein content data in the skin sample of the subject received by the data receiving module with the ACLY protein content data in the skin of the normal person consistent with the age of the subject stored in the data storage module;
(4) A judging 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, judge whether the skin physiological age of the subject accords with the actual age of the subject, and output the judgment result.
CN202110469444.XA 2021-04-28 2021-04-28 Skin rejuvenation protein marker ACLY protein and noninvasive extraction method thereof Pending CN116144621A (en)

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