Internal standard protein of skin surface lipid specimens

Abstract

To provide an internal standard protein used for quantifying a target protein contained in a skin surface lipid sample, and a method for quantifying a target protein contained in a skin surface lipid sample using the internal standard protein. [Solution] A method for quantifying the expression level of a target protein contained in a lipid sample from the skin surface, comprising using at least one protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), keratin, type I cytoskeletal 14 (KRT14), and keratin, type I cytoskeletal 10 (KRT10) as an internal standard protein.

Description

Technical Field

[0001] The present invention relates to an internal standard protein in a skin surface lipid specimen and a method for quantifying the protein expression level using the same.

Background Art

[0002] For the expression analysis of a target protein contained in a biological sample (specimen) such as cells, tissues, blood, etc., analysis methods such as Western blot, protein chip analysis, immunoassay (e.g., ELISA, etc.), immunochromatography, lateral flow immunoassay, mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), one-hybrid method, two-hybrid method, etc. are used. Generally, Western blot is widely used in protein expression analysis experiments, but an internal standard protein (endogenous control, internal control, loading control) is required for accurate relative quantitative analysis. Usually, when analyzing by Western blot, after making the total protein amount and liquid volume in the specimen constant, it is subjected to electrophoresis, and further, the expression of the internal standard protein is analyzed together with the target protein in the specimen. Thereby, it is possible to confirm whether there are any problems in the experimental operation, and by correcting the expression level of the target protein with the expression level of the internal standard protein, more accurate relative quantitative expression analysis becomes possible. Also, when analyzing by mass spectrometry, after making the total protein amount and liquid volume in the specimen constant, it is subjected to a mass spectrometer, and by correcting the expression level of the target protein with the expression level of the internal standard protein, more accurate relative quantitative expression analysis becomes possible. Also, a method for analyzing the expression level of a target protein in a specimen by immunochromatography is used for examinations and the like. In this case, usually, it is difficult to make the total protein amount and liquid volume in the specimen constant. However, even in immunochromatography, by analyzing the expression of the internal standard protein together with the target protein in the specimen, it is possible to confirm and correct the amount of the specimen used.

[0003] It is important that internal standard proteins are proteins that are highly expressed, consistently present, and universally available. Common internal standard proteins include Actin, cytoplasmic 1 (ACTB), Tubulin beta chain (TUBB), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Albumin (ALB) is sometimes used in liquid biological samples. However, it is known that the internal standard proteins that are stably expressed differ depending on the type of tissue or biological sample, and that even representative internal standard proteins may have different expression levels in certain tissues or biological samples due to physiological factors such as specific diseases. For example, it has been reported that GAPDH may not be suitable as an internal standard protein in some cases (Non-Patent Literature 1, 2).

[0004] Since keratins are constitutively expressed in epidermal keratinocytes, in addition to the aforementioned proteins, Keratin, type II cytoskeletal 1 (KRT1), Keratin, type I cytoskeletal 10 (KRT10), etc. can be used as internal standard proteins. However, it is also known that their expression can fluctuate depending on the state of epidermal keratinization (Non-Patent Documents 3, 4).

[0005] On the other hand, Patent Document 1 describes that skin surface lipids (SSL) contain RNA derived from the subject's skin cells, that the RNA contained in SSL is useful as a sample for analyzing gene expression in living organisms, and that marker genes for the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL. It is also known that proteins can be analyzed from SSL (Patent Document 2). However, appropriate internal standard proteins for protein expression analysis using SSL have not been identified. Furthermore, in the detection of atopic dermatitis, ACTB, TUBB, and ALB have been reported to function as marker proteins for atopic dermatitis (Patent Document 3), and even proteins that are generally known as internal standard proteins are not necessarily suitable as internal standard proteins for protein expression analysis using SSL. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Public Gazette No. 2018 / 008319 [Patent Document 2] Japanese Patent Publication No. 2020-074769 [Patent Document 3] Japanese Patent Publication No. 2021-175958 [Non-patent literature]

[0007] [Non-Patent Document 1] Michel-Reher MB et al., Naunyn Schmiedebergs Arch Pharmacol. 388(10):1119-20. (2015) [Non-Patent Document 2] Caradec J et al., Br J Cancer. 26;103(9):1475-6. (2010) [Non-Patent Document 3] Fuchs E et al., Trends Genet. 4(10):277-81. (1988) [Non-Patent Document 4] Van Erp PE et al., Am J Pathol. 135(5):865-70. (1989) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention relates to providing an internal standard protein for more accurately quantifying the expression level of a target protein contained in an SSL, and a method for quantifying the expression level of a target protein contained in an SSL using the internal standard protein. [Means for solving the problem]

[0009] The inventors, by comprehensively analyzing protein expression in SSL samples, identified three proteins that exhibit constitutive and high expression levels, with minimal expression variability between SSL samples. They found that these proteins are excellent internal standard proteins for quantifying the expression levels of target proteins contained in SSL.

[0010] In other words, the present invention relates to the following (1) to (4). (1) A method for quantifying the expression level of a target protein contained in a lipid sample from the skin surface, comprising using at least one protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), keratin, type I cytoskeletal 14 (KRT14), and keratin, type I cytoskeletal 10 (KRT10) as an internal standard protein. (2) A method for correcting the expression level of a target protein contained in a skin surface lipid sample, comprising using at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein. (3) A kit for quantifying the expression level of a target protein contained in a skin surface lipid sample used in the method described in (1), comprising an antibody that recognizes an internal standard protein. (4) Use of at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein in the quantification of the expression level of a target protein contained in a skin surface lipid sample. [Effects of the Invention]

[0011] The internal standard protein of the present invention exhibits constitutive and high expression levels in SSL samples, with minimal expression variability between SSL samples. Using this internal standard protein enables more accurate quantitative expression analysis of the target protein contained in SSL. [Modes for carrying out the invention]

[0012] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0013] The names of proteins disclosed herein follow the Gene Name or Protein Name listed on UniProt ([https: / / www.uniprot.org / ]).

[0014] In this specification, "internal standard protein" also refers to an endogenous control, internal control, or loading control, and means a protein that serves as a standard for correcting (standardizing, normalizing) the expression level of a target protein in protein expression analysis.

[0015] As used herein, "skin surface lipids (SSL)" refers to the lipid-soluble fraction present on the surface of the skin, and is sometimes referred to as sebum. Generally, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface. In this specification, SSL collected from a subject is also referred to as an SSL specimen.

[0016] As used herein, "skin" is a general term for a region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands, unless otherwise specified.

[0017] The three proteins, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Keratin, type I cytoskeletal 14 (KRT14), and Keratin, type I cytoskeletal 10 (KRT10), which are the internal standard proteins of the present invention, are proteins with constant expression and high expression levels in SSL specimens and small expression fluctuations among SSL specimens.

[0018] As shown in the examples described below, GAPDH, KRT14, and KRT10 were detected in the SSL specimens of all healthy subjects among the proteins extracted from the SSL specimens of 18 healthy subjects and 25 atopic dermatitis patients. The protein expression levels calculated by LC-MS / MS analysis were normalized by the Total peptide amount method to obtain protein quantitative values (Abundance) of 1.0×10 8It is above (1.0E+08), the coefficient of variation in expression (CV value) is less than 0.4, and furthermore, it is a protein found based on the protein expression level ratio (Fold change) between the SSL specimens of healthy individuals and the SSL specimens of atopic dermatitis patients calculated by the Summed Abundance Based method being within 1.0 ± 0.1 times. That is, GAPDH, KRT14, and KRT10 have constant expression and high expression levels in SSL specimens, with small variation in expression between SSL specimens. Moreover, the expression variation between the SSL specimens of healthy individuals and those of atopic dermatitis patients, which is an example of a typical skin disease, shows complex pathophysiology and is known to have large fluctuations in the expression of various genes and proteins related to inflammation, immunity, and keratinization, is also small. An internal standard protein is required to meet the conditions of having constant expression and high expression levels, having consistent expression between specimens, and not having expression fluctuations due to physiological factors such as specific diseases. Therefore, GAPDH, KRT14, and KRT10 that meet these conditions are excellent as internal standard proteins for protein expression analysis of SSL specimens. Therefore, the protein selected from the group consisting of GAPDH, KRT14, and KRT10 can be used as an internal standard protein (hereinafter also referred to as the internal standard protein of the present invention) when quantifying the expression level of the target protein contained in the SSL specimen.

[0019] The internal standard protein of the present invention is at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10, preferably GAPDH. As the internal standard protein of the present invention, any one protein selected from the group consisting of GAPDH, KRT14, and KRT10 may be used alone, or two or more proteins may be used in combination.

[0020] The internal standard protein of the present invention is suitably used in a method for quantifying the expression level of a target protein contained in an SSL sample. In one embodiment, the quantification method of the present invention includes correcting the expression level of the target protein in the SSL sample with the expression level of the internal standard protein in the SSL sample. Here, the corrected expression level of the target protein is expressed as a relative value to the expression level of the internal standard protein, so the quantification method of the present invention can also be called a relative quantification method for the expression level of the target protein contained in an SSL sample. In a preferred embodiment, the quantification method of the present invention includes measuring the expression level of the target protein in the SSL sample, measuring the expression level of the internal standard protein in the SSL sample, and correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. In a more preferred embodiment, the quantification method of the present invention includes extracting a protein from an SSL sample taken from a subject, measuring the expression level of the target protein in the SSL sample using the extracted protein, measuring the expression level of the internal standard protein in the SSL sample using the extracted protein, and correcting the measured expression level of the target protein with the measured expression level of the internal standard protein.

[0021] Furthermore, the internal standard protein of the present invention is suitably used in a method for correcting the expression level of a target protein contained in an SSL sample. In one embodiment, the correction method of the present invention includes correcting the expression level of a target protein in an SSL sample with the expression level of an internal standard protein in an SSL sample. In a preferred embodiment, the correction method of the present invention includes measuring the expression level of a target protein in an SSL sample, measuring the expression level of an internal standard protein in an SSL sample, and correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. In a more preferred embodiment, the correction method of the present invention includes extracting a protein from an SSL sample taken from a subject, measuring the expression level of a target protein in an SSL sample using the extracted protein, measuring the expression level of an internal standard protein in an SSL sample using the extracted protein, and correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. Hereinafter, the quantitative method and the correction method of the present invention may be collectively referred to as the method of the present invention.

[0022] In the present invention, the target protein is not particularly limited and may be any protein that can be contained in an SSL sample, and may be one type or two or more types.

[0023] Any means used for the recovery or removal of SSL from the skin can be employed to collect SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping off SSL from the skin, as described later, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it is a material that has an affinity for SSL, and examples include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include methods of absorbing SSL onto a sheet material such as oil-blotting paper or oil-blotting film, methods of adhering SSL to a glass plate or tape, and methods of scraping off and recovering SSL with a spatula or scraper. To improve the adsorption of SSL, an SSL absorbent material containing a highly lipid-soluble solvent beforehand may be used. On the other hand, since the adsorption of SSL is inhibited if the SSL absorbent material contains a highly water-soluble solvent or water, it is preferable that the content of highly water-soluble solvents or water is low. It is preferable to use the SSL absorbent material in a dry state. The skin from which SSL is collected is not particularly limited and can be any part of the body, such as the head, face, neck, trunk, hands, or feet. Areas with high sebum secretion, such as the skin of the face, are preferred.

[0024] SSL samples collected from subjects may be immediately used in the protein extraction process described later, or they may be stored for a certain period of time. If stored, it is preferable to store SSL samples under low temperature conditions as soon as possible after collection. The storage temperature conditions for SSL in this invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The storage period for SSL samples under these low temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours to 12 months, more preferably 6 months or less, for example, 1 day to 6 months, and even more preferably 3 months or less, for example, 3 days to 3 months.

[0025] For protein extraction from collected SSL, methods commonly used for protein extraction or purification from biological samples are employed, such as extraction using water, phosphate-buffered saline solution, or a solution containing Triton X-100, Tween20, etc. as a surfactant, or M-PER buffer (Thermo Fisher Scientific), MPEX PTS Reagent (GL Science), QIAzol Lysis Reagent (Qiagen), EasyPep TM Extraction methods using commercially available protein extraction reagents and kits, such as the Mini MS Sample Prep Kit (ThermoFisher Scientific), can be used.

[0026] To measure the expression levels of the target protein and the internal standard protein of the present invention in an SSL sample, methods commonly used in the art for measuring protein expression levels can be used. These methods are not particularly limited as long as the same method is used for measuring the expression levels of the target protein and the internal standard protein of the present invention, but examples include Western blotting, protein chip analysis, immunoassays (e.g., ELISA, ECLIA, CLEIA, CLIA, etc.), immunochromatography, lateral flow immunoassay, mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid methods, 2-hybrid methods, etc. Among these, immunoassays are preferred from the viewpoint of quantitative accuracy and sensitivity. Measuring protein expression levels may include measuring the protein content, measuring the protein concentration, and measuring the relative amount of the protein.

[0027] The correction of the expression level of the target protein in an SSL sample by the expression level of the internal standard protein of the present invention can be performed using correction methods commonly used in protein expression analysis. Such correction can typically be performed by dividing or subtracting the expression level of the target protein in the SSL sample by the expression level of the internal standard protein of the present invention in the SSL sample. Alternatively, such correction can be performed by visually evaluating and relatively comparing the intensity of the detected signals when measuring the expression levels of the target protein and the internal standard protein of the present invention in the SSL sample using a method (e.g., immunochromatography) in which a signal is detected by setting a cutoff value (reference value), or by calculating a score value based on a scale table.

[0028] The method of the present invention can be carried out, for example, when using Western blotting as a means of measuring the expression level of the target protein and the internal standard protein of the present invention in an SSL sample, as follows: A sample (for example, a sample containing protein extracted from an SSL sample) with a total protein amount or liquid volume adjusted to a constant amount is subjected to electrophoresis, and the separated protein is transferred to a nylon membrane or the like. The separated protein is treated with an antibody that specifically recognizes the target protein as a primary antibody, and then the primary antibody is labeled with an antibody against the primary antibody labeled with a radioisotope, fluorescent substance, or enzyme as a secondary antibody. The signal from these labeling substances or the signal generated by the enzymatic reaction is measured with a radiation detector, fluorescence detector, or the like. The signal for the internal standard protein of the present invention is measured in the same manner. By dividing or subtracting the signal for the obtained target protein by the signal for the internal standard protein of the present invention, the expression level of the target protein in the SSL sample can be corrected and quantified.

[0029] Furthermore, the antibody against the target protein or the internal standard protein of the present invention may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, polyclonal antibodies can be obtained by immunizing non-human animals such as rabbits with a protein expressed and purified in E. coli or the like according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, and then obtaining the antibodies from the serum of the immunized animals according to a conventional method. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing non-human animals such as mice with a protein expressed and purified in E. coli or the like according to a conventional method, or a partial polypeptide of the protein, and then fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0030] Alternatively, the method of the present invention can be carried out as follows when using an immunoassay, such as a sandwich ELISA, as a means of measuring the expression level of the target protein and the internal standard protein of the present invention in an SSL sample: A sample (for example, a sample containing protein extracted from an SSL sample) with a total protein amount or liquid volume adjusted to a constant amount is added to a solid phase immobilized with a capture antibody that specifically recognizes the target protein. The target protein captured by the capture antibody is labeled by acting on it with an enzyme-labeled antibody that specifically recognizes an epitope different from that of the target protein's capture antibody as a primary antibody, or, after acting on it with an antibody that specifically recognizes an epitope different from that of the target protein's capture antibody as a primary antibody, the primary antibody is labeled with an antibody against the enzyme-labeled primary antibody as a secondary antibody, and an enzymatic reaction is carried out by adding a substrate for these labeling enzymes (for example, a chromogenic substrate), and the signal derived from the enzymatic reaction product is measured with a spectrophotometer or the like. The signal for the internal standard protein of the present invention is measured in the same manner. The expression level of the target protein in the SSL sample can be corrected and quantified by dividing or subtracting the signal for the target protein by the signal for the internal standard protein of the present invention.

[0031] Alternatively, the method of the present invention can be carried out as follows when immunochromatography is used as a means of measuring the expression level of the target protein in an SSL sample and the expression level of the internal standard protein of the present invention: A sample (for example, a sample containing protein extracted from an SSL sample) is dropped onto the membrane of an immunochromatography kit. The signal of the test line, which has changed color due to the reaction between an antibody that specifically recognizes the target protein labeled with a metal colloid or the like and an antibody that specifically recognizes the target protein pre-captured on the membrane, is evaluated visually, scored based on a scale table, or measured using an immunochromatograph reader or other instrument to measure absorbance or fluorescence intensity. The signal for the internal standard protein of the present invention is measured in the same manner. The expression level of the target protein in the SSL sample can be corrected and quantified by visually comparing the signal for the obtained target protein and the signal for the internal standard protein of the present invention relatively, or by calculating a correction value based on a score value based on a scale table or a measurement value detected by an instrument.

[0032] Alternatively, the method of the present invention can be carried out as follows when using mass spectrometry as a means of measuring the expression level of the target protein and the expression level of the internal standard protein of the present invention in an SSL sample: A sample (for example, a sample containing protein extracted from an SSL sample) with the total protein amount or liquid volume adjusted to a constant amount is subjected to a mass spectrometer, and measurement values ​​for the target protein and the internal standard protein of the present invention are obtained by a general measurement procedure. The expression level of the target protein in the SSL sample can be corrected and quantified by dividing or subtracting the obtained measurement value for the target protein by the measurement value for the internal standard protein of the present invention.

[0033] The quantitative analysis kit for the expression level of the target protein contained in an SSL sample used in the quantitative analysis method of the present invention contains a measurement reagent for measuring the expression level of the internal standard protein of the present invention, which serves as a correction standard in SSL samples taken from a subject. Specifically, this includes a reagent for measuring the expression level of the internal standard protein of the present invention, which includes an antibody that specifically recognizes the internal standard protein of the present invention. The antibody included in the kit can be obtained by known methods as described above. In addition to the antibody mentioned above, the kit may also include a measurement reagent for measuring the expression level of the target protein, specifically a reagent for measuring the expression level of the target protein containing an antibody that specifically recognizes the target protein, a labeling reagent, a buffer, a chromogenic substrate, a secondary antibody, a blocking agent, and other equipment and controls necessary for the test, tools for collecting SSL (e.g., oil-absorbing film for collecting SSL), reagents for storing the collected SSL, a storage container, and reagents for extracting proteins from the collected SSL.

[0034] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A method for quantifying the expression level of a target protein contained in a skin surface lipid sample, comprising using at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein. <2> This includes correcting the expression level of the target protein in a skin surface lipid sample with the expression level of an internal standard protein in the skin surface lipid sample. <1> The method. <3> To measure the expression level of the target protein in a lipid sample from the skin surface; Measuring the expression level of internal standard proteins in skin surface lipid samples; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <1> The method. <4> Extracting proteins from skin surface lipid samples taken from a subject; To measure the expression level of the target protein in a skin surface lipid sample using the extracted protein; To measure the expression level of internal standard proteins in skin surface lipid samples using extracted proteins; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <1> The method. <5> The expression level of the target protein in the skin surface lipid sample is corrected by dividing it by or subtracting the expression level of the internal standard protein in the skin surface lipid sample. <2> ~ <4> One of the following methods. <6> The internal standard protein is GAPDH. <1> ~ <5> One of the following methods. <7> The expression levels of the target protein and the internal standard protein are measured by Western blotting, protein chip analysis, immunoassay, immunochromatography, lateral flow immunoassay, mass spectrometry, 1-hybrid method, or 2-hybrid method. <3> ~ <6> One of the following methods. <8> A method for correcting the expression level of a target protein contained in a skin surface lipid sample, comprising using at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein. <9> This includes correcting the expression level of the target protein in a skin surface lipid sample with the expression level of an internal standard protein in the skin surface lipid sample. <8> The method. <10> To measure the expression level of the target protein in a lipid sample from the skin surface; Measuring the expression level of internal standard proteins in skin surface lipid samples; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <8> The method. <11> Extracting proteins from skin surface lipid samples taken from a subject; To measure the expression level of the target protein in a skin surface lipid sample using the extracted protein; To measure the expression level of internal standard proteins in skin surface lipid samples using extracted proteins; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <8> The method. <12> The expression level of the target protein in the skin surface lipid sample is corrected by dividing it by or subtracting the expression level of the internal standard protein in the skin surface lipid sample. <8> ~ <11> One of the following methods. <13> The internal standard protein is GAPDH. <8> ~ <12> One of the following methods. <14> The expression levels of the target protein and the internal standard protein are measured by Western blotting, protein chip analysis, immunoassay, immunochromatography, lateral flow immunoassay, mass spectrometry, 1-hybrid method, or 2-hybrid method. <10> ~ <13> One of the following methods. <15> Contains an antibody that recognizes an internal standard protein. <1> ~ <7> A kit for quantifying target proteins contained in skin surface lipid samples used in one of the following methods.

[0035] <16> Use of at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein for quantifying the expression level of a target protein contained in a skin surface lipid sample. <17> The quantitative determination of the expression level of a target protein contained in a skin surface lipid sample includes correcting the expression level of the target protein in the skin surface lipid sample with the expression level of an internal standard protein in the skin surface lipid sample. <16> Use. <18> The quantitative determination of the expression level of the target protein contained in a skin surface lipid sample is To measure the expression level of the target protein in a lipid sample from the skin surface; Measuring the expression level of internal standard proteins in skin surface lipid samples; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <16> Use. <19> The quantitative determination of the expression level of the target protein contained in a skin surface lipid sample is Extracting proteins from skin surface lipid samples taken from a subject; To measure the expression level of the target protein in a skin surface lipid sample using the extracted protein; To measure the expression level of internal standard proteins in skin surface lipid samples using extracted proteins; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. including, <16> Use. <20> The expression level of the target protein in the skin surface lipid sample is corrected by dividing it by or subtracting the expression level of the internal standard protein in the skin surface lipid sample. <17> ~ <19> Use of either of the following. <21> The internal standard protein is GAPDH. <16> ~ <20> Use of either of the following. <22> The expression levels of the target protein and the internal standard protein are measured by Western blotting, protein chip analysis, immunoassay, immunochromatography, lateral flow immunoassay, mass spectrometry, 1-hybrid method, or 2-hybrid method. <18> ~ <21> Use of either of the following. [Examples]

[0036] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0037] Example 1: Selection of an internal standard protein using adult SSL-derived protein 1) Subjects and SSL collection This study was conducted under the approval of the Human Clinical Trials Research Ethics Committee of Kao Corporation. Eighteen healthy individuals (20-59 years old, male) and 25 patients with atopic dermatitis (20-59 years old, male) who provided informed consent were included as subjects. Skin surface lipids (SSL) were collected as sebum samples from the entire face of each subject using one oil-absorbing film (5 x 8 cm, polypropylene, Hakugen Earth). The oil-absorbing films were stored at -80°C until use for protein analysis.

[0038] 2) Preparation of sample peptide solution from sebum specimen The oil-absorbing film from which the sebum sample was collected in step 1) above was cut to an appropriate size, and a protein precipitate was obtained using QIAzol Lysis Reagent (Qiagen) according to the provided protocol. A sample peptide solution was obtained using the EasyPep Mini MS Sample Prep Kit (ThermoFisher Scientific) according to the provided protocol. TM Following the protocol of the Quantitative Fluorometric Peptide Assay (ThermoFisher Scientific), the peptide concentration in the solution was measured using a microplate reader (Corona Electric).

[0039] 3) LC-MS / MS analysis and data analysis The sample peptide solution obtained in step 2) above was subjected to LC-MS / MS analysis under the conditions shown in Table 1 below. The amount of peptide used for analysis was 120 ng.

[0040] [Table 1]

[0041] 4) Analysis method For the analysis of spectral data obtained by LC-MS / MS analysis, Proteome Discoverer ver.2.2 (ThermoFisher Scientific) was used. For protein identification, the reference database was set to Swiss Prot, the taxonomy to Homo sapiens, and a search was performed using Mascot database search (Matrix Science). In the search, Enzyme was set to Trypsin, Missed cleavage to 2, Dynamic modifications to Oxidation (M), Acetyl (N-term), Acetyl (Protein N-term), and Static Modifications to Carbamidomethyl (C). Peptides satisfying a False Discovery Rate (FDR) p<0.01 were targeted for the search. Label-free quantitative analysis (LFQ) based on precursor ions was performed on the identified proteins. Protein abundance was calculated from the peak intensity of the precursor ions derived from the peptide, and values ​​below the detection limit were treated as missing values. To correct for experimental bias, the protein abundance was normalized using the Total peptide amount method and defined as the protein quantification value (Abundance).

[0042] 5) Results LC-MS / MS analysis of proteins extracted from sebum samples collected from the entire faces of 18 healthy individuals and 25 patients with atopic dermatitis identified 1405 different proteins. The following analyses were performed to select a protein suitable as an internal standard from these proteins. To select proteins that are constitutively expressed and at high expression levels, we narrowed the selection down to 490 proteins detected in all samples from 18 healthy individuals, and further, selected those with an abundance of 1.0 × 10⁶ proteins. 8Thirty-one proteins with a coefficient of variation (CV) of 1.0E+08 or higher were obtained. Of these, since it is desirable for the expression of the internal standard protein to be constant, 17 proteins with a coefficient of variation (CV value) of less than 0.4 were selected. Furthermore, it is desirable that internal standard proteins do not fluctuate due to physiological factors such as specific diseases. Therefore, we confirmed whether these proteins fluctuate in atopic dermatitis, a representative example of a skin disease. Atopic dermatitis exhibits a complex pathophysiology, and it is known that the expression of various genes and proteins related to inflammation, immunity, and keratinization fluctuates significantly [Langan SM et al., Atopic dermatitis. Lancet. 396(10247):345-360.(2020), Pavel AB et al., J Am Acad Dermatol. 82(3):690-699. (2020)]. For the expression levels of 17 selected proteins, the ratio of protein expression levels (Fold Change) between 18 healthy individuals (HL) and 25 atopic dermatitis patients (AD) was calculated using the Summed Abundance Based method. Significance between the two groups was assessed using the Benjamini-Hochberg method, considering multiple comparisons, and the FDR was calculated. Three proteins—GAPDH, KRT14, and KRT10—showed an expression level ratio (Fold Change) of 1.0 ± 0.1. In all cases, the FDR was 0.05 or higher, indicating no significant difference between the groups. Table 2 shows the abundance, CV value, fold change (AD / HL), and FDR of these three proteins. The results above indicate that these three proteins exhibit constitutive and high expression levels, maintaining consistent expression and showing little fluctuation even in atopic dermatitis, one of the representative skin diseases. This suggests that GAPDH, KRT14, and KRT10 can function as internal standard proteins in sebum. Furthermore, regarding ACTB, TUBB, and ALB, which are generally known as internal standard proteins, the analysis results of this example showed that ACTB and TUBB were not detected in any of the 18 healthy control subjects, and the protein quantification value (Abundance) was 1.0 × 10⁻⁶. 8 Since the value was less than (1.0E+08), and although ALB met these conditions, its coefficient of variation (CV value) was 0.4 or higher, it was shown that it is not suitable as an internal standard protein for sebum.

[0043] [Table 2]

Claims

1. A method for quantifying the expression level of a target protein contained in a skin surface lipid sample, comprising using at least one protein selected from the group consisting of Glyceraldide-3-phosphote dehydrogenese (GAPDH), Keratin, type I cytoskeletal 14 (KRT14), and Keratin, type I cytoskeletal 10 (KRT10) as an internal standard protein.

2. The method according to claim 1, comprising correcting the expression level of a target protein in a skin surface lipid sample with the expression level of an internal standard protein in a skin surface lipid sample.

3. To measure the expression level of the target protein in a lipid sample from the skin surface; Measuring the expression level of internal standard proteins in skin surface lipid samples; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. The method according to claim 1, including the method described in claim 1.

4. The method according to claim 1, wherein the internal standard protein is GAPDH.

5. A method for correcting the expression level of a target protein contained in a skin surface lipid sample, comprising using at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein.

6. The method according to claim 5, comprising correcting the expression level of a target protein in a skin surface lipid sample with the expression level of an internal standard protein in a skin surface lipid sample.

7. To measure the expression level of the target protein in a lipid sample from the skin surface; Measuring the expression level of internal standard proteins in skin surface lipid samples; and Correcting the measured expression level of the target protein with the measured expression level of the internal standard protein. The method according to claim 5, including the method described in claim 5.

8. The method according to claim 5, wherein the internal standard protein is GAPDH.

9. A kit for quantifying the expression level of a target protein contained in a skin surface lipid sample used in the method according to any one of claims 1 to 4, comprising an antibody that recognizes an internal standard protein.

10. Use of at least one protein selected from the group consisting of GAPDH, KRT14, and KRT10 as an internal standard protein in the quantification of the expression level of a target protein contained in a skin surface lipid sample.

11. The use according to claim 10, wherein the internal standard protein is GAPDH.

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