CN114252630A - Skin aging protein marker SPIT1 protein and noninvasive extraction method thereof - Google Patents

Abstract

The invention discloses a skin aging protein marker SPIT1 protein and a noninvasive extraction method thereof. The application of the SPIT1 protein in auxiliary judgment of aging degree and an extraction and detection method are disclosed, aiming at finding out the intrinsic factors causing skin aging from the root, intervening the skin aging in advance before the appearance of the skin aging, and in addition, correctly judging the aging degree of the skin and judging whether the physiological age of the aging accords with the actual age, thereby providing reference and direction for beauty treatment or medical beauty treatment.

Description

Skin aging protein marker SPIT1 protein and noninvasive extraction method thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a skin aging protein marker SPIT1 protein and a noninvasive extraction method thereof.

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.

With the improvement of living standard of people, people pay more and more attention to skin care, but usually only pay attention to the external expression of skin aging, such as wrinkle, color spot, pore thickness degree and other information, and judge the aging degree of skin, the method for judging the aging degree of skin cannot find out the internal factors causing skin aging from the root, cannot intervene skin aging in advance before the external expression of skin aging occurs, and in addition, correctly judge the aging degree of skin and judge whether the physiological age of aging accords with the actual age, and is also the precondition of cosmetology or medical cosmetology.

SPINT1(SPIT1) acts as a chaperone for HSP90AA 1. Mediates the association of the molecular chaperones HSPA8/HSC70 and HSP 90. Can be combined with molecular chaperone; can be respectively combined with Hsp70 protein, Hsp90 protein, protein C terminal and RNA. In biological processes, the cells react to interleukin 7. The enzyme and pathway databases code for P31948, the reaction set codes for R-HSA-3371497, and the HSP90 chaperone cycle for Steroid Hormone Receptors (SHR). Assist in the life process of biological activities. SPINT1, also known as type 1 HGF activator (HGFA) inhibitor (HAI-1), is a Kunitz-type serine protease inhibitor that is expressed on the surface of most epithelial cells. The SPINT1-ST14 axis can regulate inflammation, and SPINT1 levels can strictly regulate tumor-to-immune cell crosstalk. Too low a level of SPINT1 results in an imbalance in cross-talk between tumor cells and their microenvironment, thus promoting higher invasiveness of SKCM (cutaneous melanoma). In addition, the study finds that the deletion of the specific Spint1 of the intestinal tract of the mouse can induce the activation of the main inflammatory transcription factor NF-kB of the intestinal tract of the mouse and accelerate the formation of intestinal tumors. Mice lacking Spint1 showed severe growth retardation and were unable to survive on day 16 after birth. These mice are histologically characterized by hyperkeratosis of the epidermis and acanthosis, as well as underdevelopment associated with abnormal stratum corneum development. Therefore, SPINT1 is considered to play an important role in maintaining immune homeostasis, inhibiting tumor development and promoting normal skin proliferation.

There is no precedent for using the SPIT1 protein (Kunitz type protease inhibitor 1) to assist in judging the degree of skin aging.

Disclosure of Invention

A method for non-invasively extracting SPIT1 protein from skin, the method 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 SPIT1 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 was performed by USPIT1 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 SPIT1 protein is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; the substance for detecting the protein content of the SPIT1 is an orbital trap high-resolution mass spectrometer.

Preferably, the P value of the SPIT1 protein is 0.018128493.

Preferably, the method for judging the aging degree and the skin aging degree by the SPIT1 protein comprises the following steps:

1) taking a sample of the epidermal skin of a subject;

2) detecting the content of SPIT1 protein in the obtained skin sample of the subject;

3) comparing the SPIT1 protein content measured in the step 2) with the SPIT1 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 SPIT1 protein content measured in the step 2) with the SPIT1 protein content standard curve in the skin of the normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.

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 SPIT1 protein content data in a skin sample of a subject;

(2) a data storage module: the data storage module is configured to store the SPIT1 protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store the SPIT1 protein content standard curve in normal human skin of each age group;

(3) a data comparison module: the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content data in normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content standard curve in normal human skin of each age group stored in the data storage module;

(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.

The invention can assist in judging the skin aging degree by detecting the SPIT1 protein content in the skin, finds out the intrinsic factors causing the skin aging from the root, intervenes the skin aging in advance before the appearance of the skin aging, can correctly judge the skin aging degree and judge whether the aging physiological age is consistent with the actual age, and provides reference and direction for beauty treatment or medical beauty treatment. The method is simple and easy to implement, and has wide market prospect.

Drawings

FIG. 1 is a mass spectrum of the characteristic peptide fragment (KDPNQVELWGLK) of the detected (SPIT1 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 SPIT1 protein from skin, the method 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 SPIT1 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 was performed by USPIT1 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 SPIT1 protein is a mass spectrometric identification reagent, an antibody or an antigen binding fragment thereof; the substance for detecting the protein content of the SPIT1 is an orbital trap high-resolution mass spectrometer.

The P value of the SPIT1 protein is 0.018128493.

The method for judging the aging degree and the skin aging degree by the SPIT1 protein comprises the following steps:

1) taking a sample of the epidermal skin of a subject;

2) detecting the content of SPIT1 protein in the obtained skin sample of the subject;

3) comparing the SPIT1 protein content measured in the step 2) with the SPIT1 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 SPIT1 protein content measured in the step 2) with the SPIT1 protein content standard curve in the skin of the normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.

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 SPIT1 protein content data in a skin sample of a subject;

(2) a data storage module: the data storage module is configured to store the SPIT1 protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store the SPIT1 protein content standard curve in normal human skin of each age group;

(3) a data comparison module: the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content data in normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content standard curve in normal human skin of each age group stored in the data storage module;

(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.

FIG. 1 is a mass spectrum of the characteristic peptide fragment (KDPNQVELWGLK) of the SPIT1 protein obtained by detection.

Randomly sampling 11 women and 9 men of normal healthy Chinese as subjects, wherein the data of the relative content of SPIT1 protein in the skin sample are as follows:

group B youth group (number)	Age (age)	Relative content of SPIT1 protein
			1	52y (Man)	10.79955732
2	55y (Man)	11.04604589
			3	60y (woman)	10.58465889
4	65y (woman)	10.51575793
			5	65y (Man)	11.5193765
6	72y (woman)	10.82707401

As can be seen from the data in the above table, the relative amount of SPIT1 protein in the skin sample of the subject increases 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 SPIT1 in each skin sample is respectively measured, for example, to serve a group of people of 35 years old in a certain city, then firstly, the skin sample of the statistically significant normal person of 35 years old living in the city is collected, the relative content of SPIT1 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 amount of SPIT1 in the skin is measured by the same method as the threshold value, and when the amount of SPIT1 is lower than the threshold value, the physiological age of the skin of the subject is younger than the actual age; when the SPIT1 content in the skin of the subject is higher than the threshold 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 amount of SPIT1 in the skin, any method capable of determining the absolute and relative amounts 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.

In addition to the skin, the amount of SPIT1 can also be used as an index to aid in determining the overall aging level of a human.

Gene:SPIT1

Protein: SPIT1 Protein

MAPARTMARARLAPAGIPAVALWLLCTLGLQGTQAGPPPAPPGLPAGADCLNSFTAGVPGFVLDTNASVSNGATFLESPTVRRGWDCVRACCTTQNCNLALVELQPDRGEDAIAACFLINCLYEQNFVCKFAPREGFINYLTREVYRSYRQLRTQGFGGSGIPKAWAGIDLKVQPQEPLVLKDVENTDWRLLRGDTDVRVERKDPNQVELWGLKEGTYLFQLTVTSSDHPEDTANVTVTVLSTKQTEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECILACRGVQGGPLRGSSGAQATFPQGPSMERRHPVCSGTCQPTQFRCSNGCCIDSFLECDDTPNCPDASDEAACEKYTSGFDELQRIHFPSDKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQQCLESCRGISKKDVFGLRREIPIPSTGSVEMAVAVFLVICIVVVVAILGYCFFKNQRKDFHGHHHHPPPTPASSTVSTTEDTEHLVYNHTTRPL。

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 SPIT1 protein from skin, the method 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 SPIT1 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 was performed by USPIT1 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 determining SPIT1 protein according to claim 2, characterized in that: the substance for detecting the content of the SPIT1 protein is a mass spectrometric identification reagent, an antibody or an antigen binding fragment thereof; the substance for detecting the protein content of the SPIT1 is an orbital trap high-resolution mass spectrometer.

4. The method for determining SPIT1 protein according to claim 2, characterized in that: the P value of the SPIT1 protein is 0.018128493.

5. The SPIT1 protein according to claim 1, wherein: the method for judging the aging degree and the skin aging degree by the SPIT1 protein comprises the following steps:

1) taking a sample of the epidermal skin of a subject;

2) detecting the content of SPIT1 protein in the obtained skin sample of the subject;

3) comparing the SPIT1 protein content measured in the step 2) with the SPIT1 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 SPIT1 protein content measured in the step 2) with the SPIT1 protein content standard curve in the skin of the normally aged person of each age, and judging the physiological age of the skin of the subject according to the comparison result.

6. 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 SPIT1 protein content data in a skin sample of a subject;

(2) a data storage module: the data storage module is configured to store the SPIT1 protein content data in normal human skin consistent with the age group of the subject or the data storage module is configured to store the SPIT1 protein content standard curve in normal human skin of each age group;

(3) a data comparison module: the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content data in normal human skin, which is consistent with the age group of the subject, stored in the data storage module; or the data comparison module is configured to compare the SPIT1 protein content data in the skin sample of the subject received by the data receiving module with the SPIT1 protein content standard curve in normal human skin of each age group stored in the data storage module;

(4) a judgment module; the judging module is configured to receive the comparison result sent by the data comparing module, judge the comparison result, judge the skin aging degree of the subject, or judge whether the skin physiological age of the subject is consistent with the actual age of the subject, and output the judgment result.

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