A hair identification method and protein marker identified therefor

Abstract

The present invention belongs to the field of bioproteomics technology, specifically relating to a hair identification method based on 4D Label-Free technology and the protein markers used to identify hair. This identification method utilizes 4D Label-Free analysis technology to obtain differentially expressed protein markers capable of distinguishing between human axillary and pubic hair samples. The protein GPRC5D described in the present invention is a protein marker specific for axillary hair, and CTNNBIP1 is a protein marker specific for pubic hair. The hair protein markers obtained in this invention can be used to identify case-related hair samples as pubic hair, which is of great significance for the determination of criminal cases.

Description

Technical Field

[0001] The present 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 easily accessible, transportable, and easily stored, hair has become a common testing material in forensic medicine and genetics. Traditional hair identification methods rely primarily on morphological analysis. However, pubic and axillary hair share many similar characteristics when observed under a microscope, which can easily lead to subjective errors. Furthermore, hair is often intentionally damaged, making its morphological structure difficult to identify.

[0003] Hair preserves a wealth of proteomic information. The proteins in hair are primarily composed of keratin, a structural fibrous protein that provides hair with strength and toughness. It can also serve as a biological sample that characterizes the state of the body over time. Because hair contains a high concentration of cystine, it forms numerous disulfide bonds and is chemically very stable, making it one of the few biological specimens that can be preserved for long periods of 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, 4D Label Free technology provides higher analytical depth and accuracy than traditional 3D Label Free analysis technology, 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, the 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 proteins in hair samples, wherein the biomarkers are 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] A 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] A third object of the present invention is to provide a hair identification kit, which comprises reagents for detecting the expression level of biomarkers, wherein the biomarkers are 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, which can more accurately identify whether the 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 solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

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

[0018] Figure 2This is a Venn diagram showing the number of proteins identified in the axillary and pubic hair groups using 4D Label-Free technology, as well as the overlap between the proteins in the axillary and pubic hair groups, identifying shared and unique proteins between the groups. MA: axillary hair sample; MP: pubic hair sample.

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

[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 accompanying 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 embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Experimental methods without specific conditions in the embodiments are generally based on conventional conditions or 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 axillary 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] 2 mg of sample was taken out from the frozen (-80°C) state and transferred to MP shaking tube (MP Biomedicals); 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 mix; use a high-throughput tissue grinder to shake 3 times, 40s each time; lyse on ice for 30min, vortex mix every 5 minutes for 5-10s; incubate at 100℃ for 10min, cool on ice; centrifuge at 4℃12000g for 20min, 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 20min, 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 20min, 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 to a final concentration of 100 mM; add tris(2-carboxyethyl)phosphine to a final concentration of 10 mM and react at 37°C for 60 min; add iodoacetamide to a final concentration of 40 mM and react at room temperature in the dark for 40 min; add pre-cooled acetone to each tube (acetone: sample v / v = 6:1) and precipitate at -20°C for 4 h; centrifuge at 10,000 g for 20 min and 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 vacuum-pumped and re-dissolved with 0.1% trifluoroacetic acid. The peptides were desalted using an HLB solid-phase extraction column and vacuum-concentrated. The peptides were quantified using a Thermo Fisher Scientific peptide quantification kit.

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

[0038] Peptides were dissolved in mass spectrometry loading buffer (2% acetonitrile, 0.1% formic acid) at a concentration of 0.1 μg / μL. 4D label-free mass spectrometry analysis was performed using an EASY-nLC 1200 liquid chromatography system (Thermo Fisher Scientific, USA) coupled to a timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany) using the LC-MS / MS platform. Peptide samples were separated on the EASY-nLC 1200 system using a C18 column (75 μm × 25 cm, Ionoptics, USA). Mobile phase A consisted of 0.1% formic acid in water and 2% acetonitrile, while mobile phase B consisted of 0.1% formic acid in water and 80% acetonitrile. The linear gradient elution process was as follows: 3% B at 0 min; 28% B at 45 min; 44% B at 50 min; 90% B at 55 min; and 90% B at 60 min. The volume flow rate was 300 nL / min, and the analysis time was 66 min. After liquid chromatography separation, samples were analyzed by mass spectrometry using a timsTOF Pro2 high-resolution mass spectrometer (4D Label Free). The mass spectrometer scan range was set to 100–1700 m / z. Data were acquired in PASEF mode. After a primary mass spectrum was acquired, 10 secondary spectra were acquired in PASEF mode for parent ion charge numbers in the range of 0–5. The cycle window time was 1.17 seconds. A dynamic exclusion time of 24 seconds was used for the MS / MS scan to prevent repeated scanning of parent ions.

[0039] 6. Protein qualitative and quantitative analysis

[0040] The original mass spectrometry files were imported into the MaxQuant version 2.0.3.1 software system for database analysis. The Human UniProt Database was used. The false discovery rate (FDR) for 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 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, and the same below.

[0041] Example 2

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

[0043] The protein genetic markers identified above were analyzed by:

[0044] (1) Analysis of protein expression differences: The protein quantitative data were calculated using the t.test function in R language to calculate the significant P value and fold change (FC) of the differences 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 the 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 discriminative efficacy of 23 differentially expressed protein markers. The results showed that the selected differentially expressed protein markers can accurately distinguish between axillary and pubic hair samples, helping 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, taking into account the important factors such as 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 (GProtein-CoupledReceptor Class C Group 5MemberD, G protein coupled receptor family C group 5 member D) is an axillary hair-specific protein marker, which is not expressed in pubic hair; CTNNBIP1 (CTNNBIP1, catenin β interacting protein 1) is a pubic hair-specific protein marker, which 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 the 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 showed that the two protein markers finally identified had similar performance in distinguishing axillary and pubic hair samples compared to the 23 protein markers tested simultaneously, and could also be used more simply, quickly, and economically to distinguish axillary and pubic hair samples. Their further application as identification reagents or kits would be beneficial to the promotion and application of the products 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 and are not limiting. 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 is to detect the expression level of biomarker proteins in hair samples, wherein the biomarkers are GPRC5D and / or CTNNBIP1, wherein GPRC5D is used for axillary hair detection; CTNNBIP1 is used for pubic hair detection; and the control is pubic hair and / or axillary hair.

2. The hair identification method according to claim 1, characterized in that: The identification method further comprises 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 any one of claims 1 to 2, characterized in that: Western blotting, mass spectrometry, liquid chromatography-mass spectrometry or enzyme-linked immunosorbent assay were used to detect the expression level of the protein.

4. 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, wherein GPRC5D is used for axillary hair detection and CTNNBIP1 is used for pubic hair detection; the control is pubic hair and / or axillary hair.

5. The use according to claim 4, characterized in that The biomarker as a target is specifically used to detect the protein expression level of the biomarker.

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