CN113189350A

CN113189350A - HBB protein as skin rejuvenation protein marker and noninvasive extraction method thereof

Info

Publication number: CN113189350A
Application number: CN202110419792.6A
Authority: CN
Inventors: 杨森; 张学军; 张博
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-19
Filing date: 2021-04-19
Publication date: 2021-07-30
Also published as: CN113030492A; CN113030493A; CN113030491A; CN113030490A; CN113030494A; CN113138280A; CN113030495A; CN113203862A

Abstract

The invention discloses a skin rejuvenation protein marker-HBB protein and a noninvasive extraction method thereof. The invention is used for detecting the application of the substance of the hemoglobin subunit beta (HBB) content of the subject in the preparation of products; the product has the function of judging or assisting in judging skin aging or skin aging degree. The invention aims to provide a basis for the skin aging degree of subjects of different ages by detecting the content of the hemoglobin subunit beta of a subject to be tested, and to age the skin by changing the content of the hemoglobin subunit beta.

Description

HBB protein as skin rejuvenation protein marker and noninvasive extraction method thereof

Technical Field

The invention relates to a skin rejuvenation protein marker HBB protein and a noninvasive extraction method thereof, belonging to the field of molecular biology.

Background

Human skin ages naturally with age or with environmental stimuli, resulting in skin aging. Natural aging is endogenous aging, which is manifested by whitening skin, fine wrinkles, decreased elasticity, skin laxity, etc., and environmental stimulation is exogenous aging, such as photoaging caused by sun exposure. If the skin is not well maintained or deteriorates with age, dead skin adheres to the surface of the skin without falling off, thereby causing a series of problems and seriously affecting beauty.

HBB proteins (hemoglobin β chain, hemoglobin subunit β, HBB) are involved in iron ion binding and oxygen binding and are members of the globin family. Hemoglobin is a ferritin protein that transports oxygen in the blood of most vertebrates.

However, there has been no report on judgment of the degree of skin aging by judgment of HBB protein.

Disclosure of Invention

The invention aims to provide a skin rejuvenation protein marker HBB protein and a noninvasive extraction method thereof.

The application of the hemoglobin subunit beta (HBB) in the auxiliary judgment of the aging degree.

In the invention, the amino acid sequence of the hemoglobin subunit beta (HBB) is shown as SEQ ID NO. 1.

The invention provides the application of the substance for detecting the content of the hemoglobin subunit beta (HBB) of a subject in preparing products; the product has the function of judging or assisting in judging skin aging.

The invention provides an application of a substance for detecting the content of hemoglobin subunit beta of a subject in preparing a product; the product has the function of diagnosing or assisting in diagnosing the skin aging degree.

In the above application, the product is a kit or a chip.

The invention relates to a product, which comprises a substance for detecting the content of beta of a hemoglobin subunit; the function of the product is as follows (a) or (b):

(a) judging or assisting in judging skin aging;

(b) diagnosing or aiding in the diagnosis of the degree of skin aging.

In the above product, the substance for detecting the content of the hemoglobin subunit β in the subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof.

In the above product, the apparatus for detecting the hemoglobin subunit β content of the subject is an orbitrap high resolution mass spectrometer.

In the above product, the product further comprises a carrier describing the following detection method:

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

2) detecting the content of the hemoglobin subunit beta in the obtained skin sample of the subject;

3) comparing the content of the hemoglobin subunit beta measured in the step 2) with the content value of the hemoglobin subunit beta in normal human skin of the age group, and judging the skin aging degree of the subject according to the comparison result;

or 4) comparing the content of the hemoglobin subunit beta measured in the step 2) with a standard curve of the content of the hemoglobin subunit beta in normal human skin of each age, and judging the aging degree of the skin of the subject or the physiological age of the skin according to the comparison result.

The invention further provides a data processing device system for assisting in judging the aging degree, which comprises the following modules:

(1) a data receiving module; the data receiving module is configured to receive hemoglobin subunit beta content data in a skin sample of a subject;

(2) a data storage module: the data storage module is configured to store data of hemoglobin subunit beta content in normal human skin consistent with the age of the subject or the data storage module is configured to store a standard curve of hemoglobin subunit beta content in normal human skin;

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

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

In the data processing device system, the system for assisting in judging the aging degree further comprises a substance for detecting the content of the hemoglobin subunit beta in the skin sample of the subject;

the substance for detecting the content of the hemoglobin subunit beta of the subject can be a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof.

In the data processing device system, the system for assisting in judging the aging degree further comprises an instrument for detecting the content of the hemoglobin subunit beta in the skin sample of the subject;

the instrument for detecting the content of the hemoglobin subunit beta in the skin sample of the subject can be an orbitrap high-resolution mass spectrometer.

The invention provides a method for extracting peptide fragments in hemoglobin subunit beta in a skin sample of a subject, which comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;

(2) obtaining of a dried peptide fragment sample: 1) the taped skin samples were cut into small pieces, applied with a sterile razor blade and deposited on glass plates before being transferred to a 1.5 ml centrifuge tube;

2) adding a proper amount of lysis buffer sample without SDS, adding 2mM EDTA and 1XCocktail, and then placing on ice for 5 minutes; then, 10mM DTT was added and the sample was soaked overnight;

3) following centrifugation at 25,000g at 4 ℃ for 15 minutes under centrifugal force, the supernatant was recovered and DTT treated with 10mM was left in a water bath at 56 ℃ for 1 hour;

4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample.

The invention further provides a method for measuring the relative content of the hemoglobin subunit beta in a skin sample of the epidermis of a subject based on mass spectrum, which comprises the following steps:

(1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;

4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatographic column and vacuum drying to obtain dried peptide sample;

(3) detection of

a) Redissolving the dried peptide fragment sample with mobile phase A (2% ACN, 0.1% FA), centrifuging at 20,000g for 10 min, and sampling the supernatant; separating by UHPLC; the sample was first enriched and desalted in a trap column and then connected in series with a self-contained C18 column (150 μm internal diameter, 1.8 μm column size, 25cm column length) at a flow rate of 500nl/min with the following effective gradient:

separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ for 180min, 5% of mobile phase B; the end of the nanoliter liquid phase separation is directly connected with a mass spectrometer;

b) DDA and/or DIA mass spectrometric detection

Detecting DDA mass spectrum:

ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, CA) to perform DDA (data-dependent acquisition) mode detection; setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000; the screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with charges 2+ to 7+, with intensities in excess of 10,000 peak intensity ranked first 20; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time is set to 30s, the AGC is set to: primary 3E6, secondary 1E 5;

DIA mass spectrometric detection:

ionizing the peptide segment subjected to liquid phase separation by a nanoESI source, and then entering a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DIA (data-independent acquisition) mode detection; the parameters are set as follows: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; uniformly dividing 350-1500Da into 40 windows for fragmentation and signal acquisition; the ion fragmentation mode is HCD, and fragment ions are detected in Orbitrap; the dynamic exclusion time was set to 30 s; the AGC is set as: primary 3E6, secondary 1E 5.

The invention has the following advantages:

the skin aging degree is judged by comparing the content of the hemoglobin subunit beta of the person to be detected with a standard curve, the method is simpler and more convenient, the result is accurate, and the efficiency is high; it provides basis for skin aging degree of subjects of different ages, and can rejuvenate skin by changing its content, and has guiding significance for skin management.

Drawings

FIG. 1 is a mass spectrum of a characteristic peptide fragment (VHLTPEEKSAVTALWGKVNVDEVGGEALGR) of the hemoglobin subunit beta (HBB) of the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Examples

The white-collar people with relatively close living environment and healthy body are taken as samples, and the epidermal skin samples are sampled by a noninvasive method (specifically 3M adhesive skin is used for completing sampling).

The method for measuring the protein content in the epidermal skin sample of the subject comprises the following steps:

(1) high performance liquid phase

Sampling of skin samples of the epidermis of a subject: sticking 3M glue (3M Health Care 3M Brookings from 3M medical glue paste manufacturers) to the curved side part of the forearm to eliminate light aging factors; after 1 minute, the 3M adhesive patch was gently removed to ensure that the skin of the subject was not damaged, and a taped skin sample was obtained.

Obtaining of a dried peptide fragment sample: 1) tape-like skin samples were cut into small pieces (0.5 x 0.5 cm), applied with a sterile razor blade and deposited on glass plates before transfer to a 1.5 ml centrifuge tube;

2) an appropriate amount of lysis buffer sample without SDS was added, 2mM EDTA, 1XCocktail was added, and then placed on ice for 5 minutes. Then, 10mM DTT was added and the sample was soaked overnight.

4) then treated with 55mM IAM, incubated for 45 minutes at room temperature in the dark, and centrifuged at 25,000g at 4 ℃ for 15 minutes to give the final protein solution supernatant; protein concentration was measured using the Bradford method, and extracted proteins were quality-controlled by 12% SDS-PAGE; taking 100 μ g of protein from each sample, adding trypsin and hydrolyzing at 37 deg.C for 4 hr; then adding trypsin again in the same proportion for enzymolysis for 8 hours at 37 ℃; desalting the polypeptide with Strata X chromatography column and drying under vacuum; thus obtaining the dried peptide fragment sample.

The dried peptide fragment sample was reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000g for 10 min, and the supernatant was injected. The separation was carried out by the Thermo company UltiMate 3000 UHPLC. The sample was first enriched and desalted in a trap column and then connected in series with a self-contained C18 column (150 μm internal diameter, 1.8 μm column size, 25cm column length) at a flow rate of 500nl/min with the following effective gradient:

separation: 0-5min, 5% mobile phase B (98% ACN, 0.1% FA); 5-160min, mobile phase B increased linearly from 5% to 35%; 160-170min, the mobile phase B rises from 35% to 80%; 170 ℃ 175min, 80% mobile phase B; 176 ℃ C. for 180min, 5% mobile phase B. The end of the nanoliter liquid phase separation was directly connected to the mass spectrometer.

(2) DDA mass spectrometric detection

The peptide fragments after liquid phase separation were ionized by nanoESI source and then introduced into a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DDA (data-dependent acquisition) mode detection. Setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 60,000; the initial m/z of the secondary mass spectrum is fixed to be 100; resolution 15,000. The screening conditions of the parent ions for secondary fragmentation are as follows: charge 2+ to 7+, parent ion with a peak intensity above 10,000 ranked first 20. The ion fragmentation mode was HCD and fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s. The AGC is set as: primary 3E6, secondary 1E 5.

(3) DIA mass spectrometric detection

The liquid phase separated peptide fragments are ionized by a nanoESI source and then enter a tandem mass spectrometer Q-active HF (Thermo Fisher Scientific, San Jose, Calif.) for DIA (data-independent acquisition) mode detection. Setting main parameters: the ion source voltage was set to 1.6 kV; the primary mass spectrum scanning range is 350-1500 m/z; resolution was set to 120,000; the 350-1500Da fragment is divided into 40 windows for fragmentation and signal collection. The ion fragmentation mode was HCD and fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s. The AGC is set as: primary 3E6, secondary 1E 5.

The mass spectrum of the characteristic peptide fragment (VHLTPEEKSAVTALWGKVNVDEVGGEALGR) of the hemoglobin subunit beta (HBB) determined by the above method is shown in FIG. 1.

The hemoglobin subunit β (HBB) in the skin samples of the subjects was measured according to the above method, and the relative content data based on mass spectra are shown in table 1 below:

TABLE 1 relative content of HBB in skin samples of subjects based on Mass Spectrometry

Age (year of old)	Relative content of HBB based on Mass Spectrometry
		20	16.9624016466176
24	16.3676220186094
		26	16.2743078802309
33	15.7484732280469

As can be seen from the data in table 1 above, the relative content of hemoglobin subunit β (HBB) in the skin sample of the subject decreases based on mass spectrometry with age.

In practical application, the method for assisting in judging the skin aging degree of a subject according to the relative content of hemoglobin subunit beta (HBB) based on mass spectrum is as follows:

(1) firstly, collecting a large number of skin samples of normal people with statistical significance at each age stage, and respectively determining the relative content of HBB in each skin sample based on mass spectrum;

(2) determining the relative content of HBB in a skin sample of a subject based on mass spectrometry; then carrying out (3) or (4);

(3) comparing the data of the subject in the step (2) with the content value of HBB in normal human skin of the age group based on mass spectrum, and judging the relative skin aging degree of the subject according to the comparison result;

(4) and (3) drawing a standard curve of age-relative content of HBB based on mass spectrum based on the data in the step (1), measuring relative content of HBB based on mass spectrum in the skin sample of the subject, and comparing the relative content with the standard curve to judge the skin aging degree or skin age of the subject.

In the present invention, the sequence of the hemoglobin subunit β is specifically as follows:

Gene:HBB

Protein:Hemoglobin subunit beta

the amino acid sequence is shown as SEQ ID NO.1, and the amino acid sequence is as follows:

MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTP DAVMGNPK

VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLV CVLAHHFG

KEFTPPVQAAYQKVVAGVANALAHKYH 。

sequence listing

<110> Yangsen

<120> HBB protein as a skin rejuvenation protein marker and a noninvasive extraction method thereof

<130> GNCLW211027

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 147

<212> PRT

<213> Homo sapiens

<400> 1

Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp

1 5 10 15

Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu

20 25 30

Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp

35 40 45

Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His

50 55 60

Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp

65 70 75 80

Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys

85 90 95

Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val

100 105 110

Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln

115 120 125

Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His

130 135 140

Lys Tyr His

145

Claims

1. Use of a substance for detecting the amount of hemoglobin subunit β in a subject for the manufacture of a product; the product has the function of judging or assisting in judging skin aging.

2. Use of a substance for detecting the amount of hemoglobin subunit β in a subject for the manufacture of a product; the product has the function of diagnosing or assisting in diagnosing the skin aging degree.

3. A product comprising a substance for detecting the amount of hemoglobin subunit β; the function of the product is as follows (a) or (b):

(a) judging or assisting in judging skin aging;

(b) diagnosing or aiding in the diagnosis of the degree of skin aging.

4. The product of claim 3, wherein: the product is a kit or a chip; and/or

The substance for detecting the content of the hemoglobin subunit beta in the subject is a mass spectrometric identification reagent, an antibody or an antigen-binding fragment thereof; and/or

The instrument for detecting the content of the hemoglobin subunit beta in the subject is an orbitrap high-resolution mass spectrometer.

5. The product according to claim 3 or 4, characterized in that: the product also comprises a carrier which is recorded with the following detection methods:

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

or 4) comparing the content of the hemoglobin subunit beta measured in the step 2) with a standard curve of the content of the hemoglobin subunit beta in normal human skin of each age, and judging the skin aging degree or the skin physiological age of the skin of the subject according to the comparison result.

6. A data processing device system for assisting in judging the aging degree comprises the following modules:

7. The data processing apparatus system of claim 6, wherein: the system for assisting in judging the aging degree also comprises a substance for detecting the content of the beta hemoglobin subunit in the skin sample of the subject;

the system for assisting in judging the aging degree also comprises an instrument for detecting the content of the beta hemoglobin subunit in the skin sample of the subject;

the substance for detecting the content of the hemoglobin subunit beta of the subject is a mass spectrometry identification reagent, an antibody or an antigen binding fragment thereof;

the instrument for detecting the content of the hemoglobin subunit beta in the skin sample of the subject is specifically an orbitrap high-resolution mass spectrometer.

8. The method for assisting in judging the degree of skin aging comprises the following steps:

1) sticking the adhesive on the skin surface of the forearm curved side part, and tearing off to obtain a skin sample of the epidermis of the subject;

2) detecting the hepatic globin content in the skin sample of the obtained subject;

3) comparing the content of the hemoglobin subunit beta measured in the step 2) with the content of the hemoglobin subunit beta in normal human skin with the same age of the subject, and judging the skin aging degree of the subject according to the comparison result;

or 4) comparing the content of the hemoglobin subunit beta measured in the step 2) with the standard curve of the content of the hemoglobin subunit beta in normal human skin with the same age, and judging the physiological age of the skin of the subject according to the comparison result;

determining that the physiological age of the skin of the subject is younger than the actual age, wherein the content of the hemoglobin subunit beta in the skin of the subject is lower than the content of the hemoglobin subunit beta in the skin of the normal human; the content of the hemoglobin subunit beta in the skin of the subject is higher than that in the skin of a normal person, and the physiological age of the skin of the subject is judged to be older than the actual age.

9. The method for extracting the peptide fragment from the hemoglobin subunit beta in the epidermal skin sample of the subject comprises the following steps: (1) sampling of skin samples of the epidermis of a subject: sticking 3M glue to the curved side part of the forearm; after 1 minute, gently removing the 3M adhesive patch to ensure that the skin of the subject is not damaged, and obtaining an adhesive tape-shaped skin sample;

10. A method for measuring the relative content of hemoglobin subunit beta in a skin sample of the epidermis of a subject based on mass spectrometry, comprising the steps of:

(3) detection of

b) DDA and/or DIA mass spectrometric detection

Detecting DDA mass spectrum:

DIA mass spectrometric detection: