Hair identification method and protein marker identified by hair identification method

Abstract

The invention belongs to the technical field of biological proteomics, and particularly relates to a hair identification method based on a 4D Lable Free technology and a protein marker identified by the hair identification method. According to the identification method, a 4D Lable Free analysis technology is utilized to obtain a protein marker capable of identifying differential expression of human axillary hair and pubic hair samples. The protein GPRC5D is an axillary hair specific protein marker, and the CTNNBIP1 is a pubic hair specific protein marker. The hair protein marker obtained by the invention can be used for identifying whether a case-related hair sample is pubic hair or not, which is of great significance to judgment of criminal events.

Description

Technical Field

[0001] The invention belongs to the technical field of biological proteomics, and relates to a hair identification method based on 4DLableFree technology and a protein marker identified therefrom. Background Art

[0002] As a biological sample that is easy to obtain, transport and store, hair has become one of the commonly used testing materials in forensic medicine and genetics. Traditional hair identification methods mainly rely on morphological analysis, but pubic hair and armpit hair have many similar features when observed under a microscope, and the identification person may easily make subjective errors when making identifications; in addition, hair is often deliberately damaged, making it difficult to identify the morphological structure.

[0003] Hair retains a wealth of proteomic information. The proteins in hair are mainly composed of keratin, a structural fibrous protein that provides strength and toughness to hair. It can also be used as a biological sample to characterize the state of the body over a certain period of time. Because hair contains more cystine, it forms a lot of disulfide bonds and has particularly stable chemical properties. It is one of the few types of biological specimens that can be preserved for a long time.

[0004] 4D Label Free technology adds a dimension to the traditional 3D Label Free technology, namely the measurement of ion mobility (Collision Cross Section, CCS). This technology uses the data-dependent acquisition-parallel accumulation-serial fragmentation (dda-PASEF) scanning mode, combined with ion mobility separation, to improve the utilization and accuracy of ions, which helps to distinguish peptides with the same mass-to-charge ratio but different structures, thereby improving the coverage depth, sensitivity and throughput of proteomics, and is more suitable for trace sample detection. In general, compared with the traditional 3D Label Free analysis technology, the 4D Label Free technology provides higher analysis depth and accuracy, and has more advantages in protein quantitative accuracy and detection cycle. It is particularly suitable for proteomics research that requires high sensitivity and high accuracy, especially when dealing with trace hair samples or requiring higher resolution to distinguish peptides, which is more reliable for confirming evidence. Summary of the invention

[0005] In view of this, an object of the present invention is to provide a hair identification method based on 4DLableFree technology, and protein markers identified in the method.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A hair identification method based on 4D Label Free technology, wherein the hair identification method specifically detects the expression level of biomarker protein in a hair sample, wherein the biomarker is GPRC5D and / or CTNNBIP1.

[0008] Furthermore, the hair identification method further comprises comparing the expression level of the detected biomarker protein with the protein expression level of the protein in a control.

[0009] Furthermore, the control is pubic hair and / or axillary hair.

[0010] Furthermore, Western blotting, mass spectrometry, liquid chromatography-mass spectrometry or enzyme-linked immunosorbent assay are used to detect the expression level of the protein.

[0011] The second object of the present invention is to provide a use of a biomarker as a target in the preparation of a hair identification kit, wherein the biomarker is GPRC5D and / or CTNNBIP1.

[0012] Furthermore, the biomarker as a target is specifically used to detect the protein expression level of the biomarker.

[0013] The third object of the present invention is to provide a hair identification kit, which comprises a reagent for detecting the expression level of a biomarker, wherein the biomarker is GPRC5D and / or CTNNBIP1.

[0014] Furthermore, the hair is pubic hair or axillary hair.

[0015] The beneficial effects of the present invention are as follows: the present invention uses 4D Label Free technology to screen protein markers for the identification of human axillary hair and pubic hair, and can more accurately identify whether a hair sample is an axillary hair sample or a pubic hair sample; at the same time, the information from proteomic characterization is complementary to the information from morphological examination and DNA analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 The results of SDS-PAGE electrophoresis analysis are shown.

[0018] Figure 2The figure is a Venn diagram, showing the number of proteins identified in the axillary hair group and the pubic hair group using the 4D Label Free technology, as well as the overlapping relationship between the proteins in the axillary hair and pubic hair sample groups, and identifying the common and unique proteins between the groups. Among them, MA: axillary hair sample; MP: pubic hair sample.

[0019] Figure 3 The volcano plot of the differentially expressed protein markers in the samples of the axillary hair group and the pubic hair group.

[0020] Figure 4 Figure 2 is a heat map of sample correlation based on 23 protein markers.

[0021] Figure 5 The circles represent the 95% confidence intervals for the principal component analysis results.

[0022] Figure 6 Verify the results by Western blotting. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods without specific conditions in the embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0025] Example 1

[0026] A method for identifying armpit hair and pubic hair based on 4D Label Free technology:

[0027] 1. Collect samples

[0028] The present invention collected 3 armpit hair samples and 3 pubic hair samples from 3 volunteers, and all samples were not subjected to any biochemical treatment.

[0029] 2. Extract hair protein

[0030] Take out 2 mg of sample from the frozen state (-80°C) and transfer it to an MP shaking tube (MP Biomedicals). Add an appropriate amount of extraction buffer (1% SDS, 200 mM DTT, 50 mM Tris-HCl, pH 8.8, containing protease inhibitor Halt TM Protease Inhibitor Cocktail), vortex to mix; use a high-throughput tissue grinder to shake 3 times, 40 seconds each time; lyse on ice for 30 minutes, vortex to mix for 5-10 seconds every 5 minutes; incubate at 100℃ for 10 minutes, cool on ice; centrifuge at 4℃12000g for 20 minutes, and take the supernatant; add pre-cooled acetone at a volume ratio of 1:4, and precipitate overnight at -20℃; the next day, centrifuge at 4℃12000g for 20 minutes, discard the supernatant, add 90% pre-cooled acetone to the precipitate, mix well, centrifuge and discard the supernatant, repeat 2 times; dissolve the precipitate with protein lysis buffer (8M urea + 1% SDS, containing protease inhibitors); centrifuge at 4℃, 12000g for 20 minutes, and take the protein supernatant. Then determine the protein content by BCA method (see Table 1), and then use SDS-PAGE electrophoresis analysis to detect the protein to ensure that the sample bands are clear and abundant. The electrophoresis results are as follows Figure 1 shown.

[0031] Table 1 BCA quantitative results

[0032]

[0033] 3. Proteolysis

[0034] Take 100 μg of protein sample, supplement with lysis buffer, add triethylammonium bicarbonate buffer with a final concentration of 100 mM; add tri(2-carboxyethyl)phosphine with a final concentration of 10 mM, react at 37°C for 60 min; add iodoacetamide with a final concentration of 40 mM, react at room temperature in the dark for 40 min; add pre-cooled acetone to each tube (acetone: sample v / v = 6:1), precipitate at -20°C for 4 h; centrifuge at 10000g for 20 min, take the precipitate; fully dissolve the sample with 100 μL 100 mM triethylammonium bicarbonate buffer; add Trypsin at an enzyme: protein (m / m) ratio of 1:50 and enzymatically hydrolyze at 37°C overnight.

[0035] 4. Peptide desalting and quantification

[0036] After trypsin digestion, the peptides were dried with a vacuum pump; the peptides after enzymatic digestion and drying were re-dissolved with 0.1% trifluoroacetic acid; the peptides were desalted with an HLB solid phase extraction column and dried with a vacuum concentrator; and the peptides were quantified using the Thermo Fisher Scientific Peptide Quantification Kit.

[0037] 5. 4D Label Free Technology Analysis Based on LC-MS / MS Platform

[0038] After dissolving peptides at a concentration of 0.1 μg / μL in mass spectrometry loading buffer (2% acetonitrile 0.1% formic acid), 4D Label Free mass spectrometry analysis was performed using the LC-MS / MS platform of the EASY-nLC 1200 liquid chromatography system (Thermo Fisher Scientific, USA) coupled to the timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany). The peptide samples were separated by the EASY-nLC1200 liquid chromatography system, and the chromatographic column was a C18 column (75 μm×25 cm, Ionopticks, USA). Mobile phase A consisted of 0.1% formic acid aqueous solution and 2% acetonitrile, while mobile phase B consisted of 0.1% formic acid aqueous solution and 80% acetonitrile. The linear gradient elution process was 0 min, 3% B; 45 min, 28% B; 50 min, 44% B; 55 min, 90% B; 60 min, 90% B. The volume flow rate was 300 nL / min and the analysis time was 66 min. The samples separated by liquid chromatography were analyzed by mass spectrometry using a timsTOF Pro2 high-resolution mass spectrometer (4D Lable Free). The mass spectrometry scanning range was set to 100-1700 m / z. The data acquisition used the PASEF mode. After one primary mass spectrum acquisition, 10 PASEF modes were used to acquire the secondary spectra with parent ion charge numbers in the range of 0-5. The cycle window time was 1.17 seconds. The dynamic exclusion time of the MS / MS scan was 24 seconds to avoid repeated scanning of the parent ion.

[0039] 6. Protein qualitative and quantitative analysis

[0040] The original files from the mass spectrometer were imported into the MaxQuant version 2.0.3.1 software system for database search and analysis, and the database used was the Human UniProt Database. The false discovery rate (FDR) of peptide identification was set to ≤0.01. The identified proteins contained at least one specific peptide. The present invention identified 547 and 565 protein markers in axillary hair and pubic hair samples, respectively. Figure 2 The Venn diagram shows the number of proteins in the axillary hair group and pubic hair group samples and the overlapping relationship of proteins between the groups, identifying the common and unique proteins between the groups, where MA: axillary hair sample; MP: pubic hair sample, the same below.

[0041] Example 2

[0042] Screening of protein markers for identification of axillary and pubic hair

[0043] The protein genetic markers identified above were:

[0044] (1) Analysis of protein expression differences: The protein quantitative data were calculated using the t.test function in the R language to calculate the significant P value and fold change (FC) between groups. Proteins with a significance test p<0.05 and a fold change>2.0 were up-regulated proteins, and proteins with a p<0.05 and a fold change<0.5 were down-regulated proteins. The present invention retains a total of 23 differentially expressed protein markers for identification of axillary hair and pubic hair. Figure 3 The difference volcano plots of the differentially expressed protein sets in the axillary and pubic hair group samples are shown.

[0045] (2) Expression pattern cluster analysis:

[0046] Furthermore, the present invention adopts a sample correlation heat map ( Figure 4 , where asterisks represent the statistical significance level of the correlation between samples, and the more asterisks, the more significant the correlation) and principal component analysis ( Figure 5 ) method to evaluate the identification efficiency of 23 differentially expressed protein markers. The results showed that the screened differentially expressed protein markers can accurately identify axillary and pubic hair samples, which helps to determine the relevance of hair samples to the case and determine the nature of the case.

[0047] (3) Re-screening of 23 protein markers:

[0048] Furthermore, considering the convenience of identification, low identification cost and product promotion cost in practical applications of the hair identification method, the present invention finally chose to retain two specifically expressed protein markers. GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D) is an axillary hair-specific protein marker that is not expressed in pubic hair; CTNNBIP1 (CTNNBIP1, beta-catenin β interacting protein 1) is a pubic hair-specific protein marker that is not expressed in axillary hair. The expression levels of these two protein markers in pubic hair and axillary hair in Example 1 and the results of differential analysis are shown in Table 2. Two independent samples from two different volunteers (2 each of pubic hair and axillary hair) were taken for verification (sample numbers MA4, MA5, MP4, MP5), and the Western blotting verification results of these two protein markers are shown in Table 2. Figure 6The results show that the two protein markers finally determined, compared with the 23 protein markers detected simultaneously, have similar performance in identifying axillary hair and pubic hair samples, and can identify axillary hair and pubic hair samples more simply, quickly and economically, and can be further used as identification reagents or kits, which is conducive to the promotion and application of the product of the present invention.

[0049] The GPRC5D protein sequence is as follows:

[0050] 1mykdciestg dyfllcdaeg pwgiilesla ilgivvtill llaflflmrk iqdcsqwnvl

[0051] 61ptqllfllsv lglfglafafiielnqqtap vryflfgvlfalcfscllah asnlvklvrg

[0052] 121cvsfswttil ciaigcsllq iiiateyvtl imtrgmmfvn mtpcqlnvdfvvllvyvlfl

[0053] 181maltffvska tfcgpcenwk qhgrlifitv lfsiiiwvvw ismllrgnpq fqrqpqwddp

[0054] 241vvcialvtna wvflllyivp elcilyrscr qecplqgnac pvtayqhsfq venqelsrdc

[0055] The CTNNBIP1 protein sequence is as follows:

[0056] 1mnregapgks peemyiqqkv rvllmlrkmg snltaseeeflrtyagvvns qlsqlpphsi

[0057] 61dqgaedvvma fsrsetedrr q

[0058] Table 2 Expression levels and differential analysis results of protein markers GPRC5D and CTNNBIP1 in pubic hair and axillary hair

[0059]

[0060] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A hair identification method, characterized in that: The hair identification method specifically detects the expression level of biomarker proteins in hair samples, and the biomarkers are GPRC5D and / or CTNNBIP1.

2. The hair identification method according to claim 1, characterized in that: The identification method also includes comparing the expression level of the detected biomarker protein with the protein expression level of the protein in a control.

3. The hair identification method according to claim 2, characterized in that: The control is pubic hair and / or axillary hair.

4. The hair identification method according to any one of claims 1 to 3, characterized in that: The expression level of the protein can be detected using Western blotting, mass spectrometry, liquid chromatography-mass spectrometry or enzyme-linked immunosorbent assay.

5. Use of a biomarker as a target in the preparation of a hair identification kit, characterized in that: The biomarkers are GPRC5D and / or CTNNBIP1.

6. The use according to claim 5, characterized in that: The biomarker as a target is specifically used to detect the protein expression level of the biomarker.

7. A hair identification kit, characterized in that: The hair identification kit comprises reagents for detecting the expression level of biomarkers, and the biomarkers are GPRC5D and / or CTNNBIP1.

8. The hair identification kit according to claim 7, characterized in that: The hair is pubic hair or axillary hair.

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