A rapid pre-treatment method for hair shaft proteome samples
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-11-28
- Publication Date
- 2026-05-29
Smart Images
Figure CN122109400A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a rapid pretreatment method for hair shaft proteome samples, belonging to the field of proteome analysis technology. Background Technology
[0002] Hair shafts are among the most common biological evidence found at crime scenes, characterized by a long detection window, ease of collection, and convenient preservation (Forensic Sci Int Genet, 2023, 66:1-13). The apoptosis of keratinocytes during the later stages of hair shaft formation is accompanied by the breakage and degradation of nuclear DNA, resulting in low DNA content and severe fragmentation (AnnAnat, 2021, 194(1):31-35). This significantly reduces the feasibility of individual identification through single nucleotide polymorphism (SNP) site detection in hair shaft DNA. Hair shafts are rich in protein, and the numerous disulfide bonds between proteins make them chemically more stable. They also contain a large number of single amino acid polymorphism (SAP) sites translated from non-synonymy SNPs (nsSNPs) in the genome. Combining proteomics analysis to obtain SAP sites for individual identification presents an attractive alternative for forensic identification. The hair shaft consists of three layers: the medulla is the innermost layer, the cortex is the middle layer, and the outermost layer is the squamous layer. Studies have shown that the main hair shaft proteins, keratin (KRT) and keratin-associated protein (KAP), are specifically expressed in each layer of the hair shaft. Due to its high keratin content and the presence of a large number of transglutaminase-mediated heteropeptide crosslinks, about 15% of the constituent proteins are difficult to dissolve even under strong denaturing agents (PLoS One, 2016, 11(10), e0164993). It is usually necessary to add dissolving agents, surfactants, etc. to aid solubilization during protein extraction. Due to the incompatibility between solubilizers and proteases, it is usually necessary to remove the solubilizers for a long time before enzymatic digestion. In the process of proteomics analysis, the interference of high-abundance proteins such as keratin reduces the resolution of low-abundance proteins, thus limiting the identification coverage. In addition, the amount of hair shaft obtained at crime scenes is often limited. Therefore, it is urgent to develop a rapid proteomics sample pretreatment method with high recovery rate and high sensitivity suitable for trace hair shaft samples.
[0003] To address the aforementioned issues, this invention provides a rapid pretreatment method for hair shaft proteomics samples. This method enables high-recovery protein extraction, rapid enzymatic digestion, and high-sensitivity analysis of trace hair shaft samples using liquid chromatography-mass spectrometry (LC-MS), providing an important tool for the application of hair shaft proteomics in forensic medicine and other fields. Summary of the Invention
[0004] This invention provides a rapid pretreatment method for hair shaft proteomic samples. A mixed solution of guanidine hydrochloride, 1-dodecyl-3-methylimidazole chloride (C12 Im-Cl), and tris(2-carboxyethyl)phosphine (TCEP) is used as the extraction reagent. The hair shaft is broken up using ultrasonic energy and shear force to extract the hair shaft protein. After denaturation and reduction, the protein is enzymatically digested on a filter membrane. A precipitant is added before enzymatic digestion to reduce the number of washing steps and shorten the pretreatment time. Finally, a protease is added for membrane digestion. After centrifugation, a peptide solution is obtained for proteomic analysis using liquid chromatography-mass spectrometry (LC-MS). This pretreatment method requires a small amount of hair shaft sample, has a short operation time, and provides high protein identification coverage, meeting the needs of forensic medicine and other fields for high-throughput analysis of trace hair shaft samples.
[0005] The technical solution of this invention is as follows:
[0006] The hair shaft was ultrasonically washed with methanol / water solution to remove the washing solution;
[0007] The extraction reagent was a mixed solution of guanidine hydrochloride, 1-dodecyl-3-methylimidazole chloride (C12 Im-Cl), and tris(2-carboxyethyl)phosphine (TCEP). Sodium hydroxide solution was added to the extraction reagent to adjust the pH to 7-8. The solution was then added to the hair sample, heated, shaken, and soaked. Subsequently, the hair was broken up using ultrasonic energy and shear force.
[0008] Centrifuge the broken hair shaft suspension and take the supernatant as the hair shaft protein extraction solution. Add tris(2-carboxyethyl)phosphine (TCEP) solution to the hair shaft protein extraction solution to further denature and reduce the protein.
[0009] The solution was transferred to a filter membrane with a molecular weight cutoff of 2-20 kDa, the waste liquid was removed by centrifugation, an alkylating agent was added to the filter membrane to carry out the alkylation reaction, the reaction solution was removed by centrifugation, and the filter membrane was cleaned by adding a cleaning solution.
[0010] Add the extraction reagent precipitant to the filter membrane to reduce the number of times the extraction reagent needs to be washed. Add the precipitant dropwise at a rate of 1-10 μL / sec, and incubate with shaking at 20℃-35℃ for 1-10 minutes. Centrifuge to remove the waste liquid.
[0011] Add cleaning solution to the filter membrane, centrifuge to clean the filter membrane, and repeat this operation 1-20 times;
[0012] Add washing solution and protease to the filter membrane, hydrolyze at 37°C, and centrifuge. The filtrate is the peptide solution.
[0013] The peptide solution was frozen for detection using liquid chromatography-mass spectrometry.
[0014] The liquid chromatography conditions are as follows:
[0015] The chromatographic column is a reversed-phase capillary column with an inner diameter of 50-150 micrometers and a length of 5-30 centimeters;
[0016] The mobile phase consists of mobile phase A and mobile phase B in a certain proportion for gradient elution;
[0017] Mobile phase A is acetonitrile with a volume fraction of 0% to 2% (containing at least one of formic acid, acetic acid, and trifluoroacetic acid with a volume fraction of 0.01% to 0.5%);
[0018] Mobile phase B is 80%-100% acetonitrile (containing at least one of formic acid, acetic acid, and trifluoroacetic acid, with a volume fraction of 0.01%-0.5%).
[0019] The flow rate of the mobile phase is 100-1000 nanoliters per minute;
[0020] The mass spectrometry conditions were as follows: at least one of the following was used: electrospray ionization source, atmospheric pressure chemical ionization source or matrix-assisted laser desorption / ionization source, positive ion mode, and at least one of the following acquisition methods: data-dependent or data-independent.
[0021] The present invention has the following advantages:
[0022] 1. The extraction reagent can effectively break disulfide bonds and dissolve hair shaft protein, thereby improving the extraction efficiency of hair shaft protein;
[0023] 2. The addition of precipitant to the extraction reagent can shorten the number of times the extraction reagent needs to be washed and make the washing of the extraction reagent more thorough, avoiding the influence of the extraction reagent on the enzymatic hydrolysis process, thereby improving the enzymatic hydrolysis efficiency, shortening the enzymatic hydrolysis time, and improving the identification coverage;
[0024] 3. The combination of high protein extraction efficiency and high enzymatic hydrolysis efficiency allows for the use of hair shafts down to the millimeter level. The rapid extraction reagent cleaning process and improved enzymatic hydrolysis efficiency can shorten the detection time, thereby meeting the needs of forensic and other fields for rapid, high-sensitivity, and high-throughput proteomics detection with low sample volumes. Attached Figure Description
[0025] Figure 1 This is the base peak diagram for mass spectrometry analysis of sample number 001.
[0026] Figure 2 This is the base peak diagram for mass spectrometry analysis of sample number 002.
[0027] Figure 3 This is the base peak diagram for mass spectrometry analysis of sample number 003.
[0028] Figure 4This is the base peak diagram for mass spectrometry analysis of sample number 004.
[0029] Figure 5 This is the base peak diagram for mass spectrometry analysis of sample number 005.
[0030] Figure 6 This is the base peak diagram for mass spectrometry analysis of sample number 006.
[0031] Figure 7 This is the base peak diagram for mass spectrometry analysis of sample number 007.
[0032] Figure 8 This is the base peak diagram for mass spectrometry analysis of sample number 008.
[0033] Figure 9 This is the base peak diagram for mass spectrometry analysis of sample number 009. Detailed Implementation
[0034] The present invention will now be described in detail with reference to the embodiments, but the present invention is not limited to these embodiments.
[0035] Unless otherwise specified, the raw materials used in the embodiments of the present invention were all purchased commercially.
[0036] Example 1
[0037] 1. Preparation of extraction reagent: Mix 6M guanidine hydrochloride and 10% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 3:14, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 400mM;
[0038] 2. Wash a 0.5 cm long hair shaft (number 001, human hair) with a 40% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 100 μL of extraction reagent (0.3 mg hair shaft / mL extraction reagent). Add 100 μL of 0.5 M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 37°C with shaking for 2 hours to soften it. Use contact sonication for 15 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0039] 3. Add 100 μL of 100 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.5) to the supernatant, denature and reduce at 95°C for 5 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM iodoacetamide solution to the filter membrane, incubate at room temperature in the dark for 10 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 100 mM phosphate (PBS) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0040] 4. Add 50 μL of 50% lithium hexafluorophosphate solution to the filter membrane at a rate of 3 μL / s, incubate with shaking at 30°C for 1 minute to precipitate and extract the reagent to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid;
[0041] 5. Add 100 μL of 100 mM PBS buffer solution to the filter membrane and centrifuge at 20,000 g to wash the filter membrane. Repeat this washing operation 4 times.
[0042] 6. Add 100 μL of 100 mM PBS buffer solution to the filter membrane and add 1 μg of Lys-C enzyme (protease to hair protein mass ratio 1:12). Incubate at 37°C for 5 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0043] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0044] The chromatographic system was EASY-nano LCTM1000;
[0045] The chromatographic column is a C18 reversed-phase capillary column with an inner diameter of 150 micrometers and a length of 20 centimeters;
[0046] The mobile phase consists of mobile phase A and mobile phase B in a certain proportion for gradient elution;
[0047] Mobile phase A is 2% acetonitrile (containing 0.1% formic acid by volume);
[0048] Mobile phase B is 80% acetonitrile (containing 0.1% formic acid by volume);
[0049] The flow rate of the mobile phase is 600 nanoliters per minute;
[0050] The gradient (V / V) was as follows: 0-29 min, 12%-30% B; 29-32 min, 30%-38% B; 32-34 min, 38%-95% B; 34-40 min, 95% B.
[0051] Mass spectrometry conditions are:
[0052] The mass spectrometry system was an Orbitrap Exploris 480.
[0053] Data acquisition was performed using a data-dependent mode. The Full MS scan range was 350-1500 m / z with a resolution of 60000. The normalized automatic gain control (AGC) target was set to 300%, and the maximum injection time (IT) was 20 ms. The MSMS parameters were: resolution 15000, normalized AGC target set to 300%, IT 30 ms, separation window 1.6 m / z, fragmentation mode High-energy collision dissociation (HCD), normalized collision energy 30%, and FAIMS compensation voltage (CV) -45V and -65V.
[0054] As a result, the recovery rate of sample 001 was 74.8%, and 4862 peptides and 852 proteins were identified.
[0055] Example 2
[0056] 1. Preparation of extraction reagent: Mix 8M guanidine hydrochloride and 10% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 1:8, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 300mM;
[0057] 2. Wash an 8 cm long hair shaft (number 002, human hair) with a 60% methanol solution for 5 minutes using ultrasonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 200 μL of extraction reagent (2.4 mg hair shaft / mL extraction reagent). Add 100 μL of 1M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 37°C with shaking for 3 hours to soften it. Use contact sonication for 40 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0058] 3. Add 100 μL of 100 mM TCEP (3:1 volume ratio of hair dry protein solution to TCEP solution) to the supernatant, denature and reduce at 56°C for 15 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM chloroacetamide solution to the filter membrane, incubate at room temperature for 20 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 50 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, centrifuge at 20000g to wash the filter membrane;
[0059] 4. Add 100 μL of 40% lithium bis(trifluoromethane)sulfonylimide solution to the filter membrane at a rate of 10 μL / s, incubate at 23°C with shaking for 5 minutes to precipitate and extract the reagent to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0060] 5. Add 100 μL of 50 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to clean the filter membrane. Repeat this cleaning operation 4 times.
[0061] 6. Add 100 μL of 50 mM ABC buffer solution to the filter membrane and add 15 μg of trypsin (protein to hair protein mass ratio 1:13). Incubate at 37°C for 3 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0062] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0063] The liquid chromatography and mass spectrometry conditions were the same as in Example 1.
[0064] As a result, the recovery rate of sample 002 was 63.0%, and 6218 peptides and 1305 proteins were identified.
[0065] Example 3
[0066] 1. Preparation of extraction reagent: Mix 6M guanidine hydrochloride and 12% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 1:6, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 200mM;
[0067] 2. Wash a 1 cm long hair shaft (number 003, human hair) with an 80% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 100 μL of extraction reagent (0.6 mg hair shaft / mL extraction reagent). Add 100 μL of 0.5 M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 50°C with shaking for 1 hour to soften it. Use contact sonication for 20 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0068] 3. Add 100 μL of 300 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.5) to the supernatant, denature and reduce at 95°C for 5 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM iodoacetamide solution to the filter membrane, incubate at room temperature in the dark for 20 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 50 mM phosphate (PBS) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0069] 4. Add 50 μL of 50% lithium bis(trifluoromethanesulfonyl)imide solution to the filter membrane at a rate of 5 μL / s, shake and incubate at 30°C for 2 minutes to precipitate and extract the reagent to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0070] 5. Add 100 μL of 50 mM PBS buffer solution to the filter membrane and centrifuge at 20,000 g to wash the filter membrane. Repeat this washing operation 6 times.
[0071] 6. Add 100 μL of 50 mM PBS buffer solution to the filter membrane, and add 5 μg of Lys-C enzyme (protease to hair protein mass ratio 1:5). Incubate at 37°C for 1 hour. After centrifugation at 20000g, the filtrate is the peptide solution.
[0072] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0073] The chromatographic system was EASY-nano LCTM1000;
[0074] The chromatographic column is a C18 reversed-phase capillary column with an inner diameter of 150 micrometers and a length of 20 centimeters;
[0075] The mobile phase consists of mobile phase A and mobile phase B in a certain proportion for gradient elution;
[0076] Mobile phase A is 2% acetonitrile (containing 0.1% formic acid by volume);
[0077] Mobile phase B is 80% acetonitrile (containing 0.1% formic acid by volume);
[0078] The flow rate of the mobile phase is 600 nanoliters per minute;
[0079] The gradient (V / V) was as follows: 0-45 min, 12%-30% B; 45-51 min, 30%-38% B; 51-53 min, 38%-95% B; 53-60 min, 95% B.
[0080] Mass spectrometry conditions are:
[0081] The mass spectrometry system was an Orbitrap Exploris 480.
[0082] Data acquisition was performed using a data-dependent mode. The Full MS scan range was 350-1500 m / z with a resolution of 60000. The normalized automatic gain control (AGC) target was set to 300%, and the maximum injection time (IT) was 20 ms. The MSMS parameters were: resolution 15000, normalized AGC target set to 300%, IT 30 ms, separation window 1.6 m / z, fragmentation mode High-energy collision dissociation (HCD), normalized collision energy 30%, and FAIMS compensation voltage (CV) -45V and -65V.
[0083] As a result, the recovery rate of sample 003 was 69.7%, and 5208 peptides and 905 proteins were identified.
[0084] Example 4
[0085] 1. Preparation of extraction reagent: Mix 6M guanidine hydrochloride and 10% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 3:17, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 200mM;
[0086] 2. Wash a 2 cm long hair shaft (number 004, human hair) with a 50% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 100 μL of extraction reagent (1.2 mg hair shaft / mL extraction reagent). Add 100 μL of 0.5 M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 37°C with shaking for 3 hours to soften it. Use contact sonication for 20 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0087] 3. Add 100 μL of 200 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.5) to the supernatant, denature and reduce at 95°C for 5 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM chloroacetamide solution to the filter membrane, incubate at room temperature for 20 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 20 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0088] 4. Add 50 μL of 40% lithium bis(trifluoromethane)sulfonylimide solution to the filter membrane at a rate of 8 μL / s, shake and incubate at 35°C for 1 minute to precipitate and extract the reagent to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0089] 5. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to clean the filter membrane. Repeat this cleaning operation 4 times.
[0090] 6. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and add 5 μg of trypsin (protein to hair protein mass ratio 1:10). Incubate at 37°C for 3 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0091] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0092] The liquid chromatography and mass spectrometry conditions were the same as in Example 3.
[0093] As a result, the recovery rate of sample 004 was 72.0%, and 5775 peptides and 1204 proteins were identified.
[0094] Example 5
[0095] 1. Preparation of extraction reagent: Mix 4M guanidine hydrochloride with 15% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 1:2, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 250mM;
[0096] 2. Wash a 3 cm long hair shaft (number 005, human hair) with a 50% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 150 μL of extraction reagent (1.2 mg hair shaft / mL extraction reagent). Add 75 μL of 1M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 50°C with shaking for 0.5 hours to soften it. Use contact sonication for 40 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0097] 3. Add 90 μL of 200 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.4) to the supernatant, denature and reduce at 60°C for 10 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM chloroacetamide solution to the filter membrane, incubate at room temperature for 20 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 20 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0098] 4. Add a 40% (w / w) solution of lithium bis(trifluoromethanesulfonyl)imide to the filter membrane at a rate of 3 μL / s, incubate with shaking at 25°C for 2 minutes to precipitate the extractant and reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0099] 5. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to clean the filter membrane. Repeat this cleaning operation 4 times.
[0100] 6. Add 100 μL of 20 mM ABC buffer solution to the filter membrane, and add 3 μg each of trypsin and Arg-C enzyme (protein to hair protein mass ratio 1:12). Incubate at 37°C for 4 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0101] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0102] The liquid chromatography and mass spectrometry conditions were the same as in Example 1.
[0103] As a result, the recovery rate of sample 005 was 68.0%, and 5901 peptides and 1220 proteins were identified.
[0104] Example 6
[0105] 1. Preparation of extraction reagent: Add tris(2-carboxyethyl)phosphine (TCEP) to a 12% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) to make the final concentration 500mM;
[0106] 2. Wash a 2 cm long hair shaft (number 006, human hair) with a 50% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 100 μL of extraction reagent (1.2 mg hair shaft / mL extraction reagent). Add 100 μL of 0.5 M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 37°C with shaking for 4 hours to soften it. Use contact sonication for 30 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0107] 3. Add 100 μL of 50 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.5) to the supernatant, denature and reduce at 95°C for 5 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM chloroacetamide solution to the filter membrane, incubate at room temperature in the dark for 15 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 30 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, centrifuge at 20000g to wash the filter membrane;
[0108] 4. Add a 50% (w / w) lithium bis(trifluoromethane)sulfonylimide solution to the filter membrane at a rate of 5 μL / s, incubate with shaking at 25°C for 2 minutes to precipitate the extract to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0109] 5. Add 100 μL of 30 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to wash the filter membrane. Repeat this washing operation 3 times.
[0110] 6. Add 100 μL of 30 mM ABC buffer solution to the filter membrane and add 10 μg of trypsin (protein to hair protein mass ratio 1:5). Incubate at 37°C for 3 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0111] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0112] The liquid chromatography and mass spectrometry conditions were the same as in Example 1.
[0113] As a result, the recovery rate of sample 006 was 38.5%, with 2330 peptides and 424 proteins identified. The main reason for the low recovery rate and lower identification coverage than in Examples 1-5 was that guanidine hydrochloride was not added to the extraction solution. Guanidine hydrochloride can destroy hydrogen bonds and hydrophobic bonds in hair shaft protein, increase protein solubility, and improve protein extraction recovery rate. Without the addition of guanidine hydrochloride, even with increased soaking and sonication time, the recovery rate and identification coverage of Examples 1-5 could not be achieved.
[0114] Example 7
[0115] 1. Preparation of extraction reagent: Add tris(2-carboxyethyl)phosphine (TCEP) to 6M guanidine hydrochloride solution to make a final concentration of 200mM;
[0116] 2. Wash a 2 cm long hair shaft (number 007, human hair) with a 50% methanol solution for 5 minutes using ultrasonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 200 μL of extraction reagent (0.6 mg hair shaft / mL extraction reagent). Add 100 μL of 1M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 50°C with shaking for 6 hours to soften it. Use contact sonication for 40 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0117] 3. Add 100 μL of 200 mM TCEP (3:1 volume ratio of hair dry protein solution to TCEP solution) to the supernatant, denature and reduce at 95°C for 5 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM iodoacetamide solution to the filter membrane, incubate at room temperature in the dark for 25 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 20 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, centrifuge at 20000g to wash the filter membrane;
[0118] 4. Add a 50% (w / w) bis(trifluoromethane)sulfonylimide lithium salt solution to the filter membrane at a rate of 5 μL / s, incubate with shaking at 25°C for 4 minutes to precipitate the extract to reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0119] 5. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to clean the filter membrane. Repeat this cleaning operation 7 times.
[0120] 6. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and add 5 μg of trypsin (protein to hair protein mass ratio 1:10). Incubate at 37°C for 6 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0121] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0122] The liquid chromatography and mass spectrometry conditions were the same as in Example 1.
[0123] As a result, the recovery rate of sample 007 was 35.7%, with 1568 peptides and 390 proteins identified. The main reason for the low recovery rate and lower identification coverage compared to Examples 1-5 was the absence of 1-dodecyl-3-methylimidazole chloride (C12 Im-Cl) in the extraction solution. C12 Im-Cl can break the disulfide bonds in the hair shaft protein, increase the solubility of the protein, and improve the protein extraction recovery rate. At the same time, the full exposure of the disulfide bonds can also improve the enzymatic hydrolysis efficiency and increase the identification coverage. Without the addition of C12 Im-Cl, even with increased soaking and sonication time, the recovery rate and identification coverage of Examples 1-5 could not be achieved.
[0124] Example 8
[0125] 1. Preparation of extraction reagent: Mix 6M guanidine hydrochloride with 15% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 3:16, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 200mM;
[0126] 2. Wash a 3 cm long hair shaft (number 008, human hair) with a 50% methanol solution for 5 minutes using ultrasonication to remove the washing liquid. Cut the hair shaft into pieces about 1 mm in size and add 150 μL of extraction reagent (1.2 mg hair shaft / mL extraction reagent). Add 75 μL of 1M sodium hydroxide solution to adjust the pH to 7.5. Use contact sonication for 90 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0127] 3. Add 90 μL of 200 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.4) to the supernatant, denature and reduce at 95°C for 10 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM chloroacetamide solution to the filter membrane, incubate at room temperature for 20 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 20 mM ammonium bicarbonate (ABC) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0128] 4. Add a 40% (w / w) solution of lithium bis(trifluoromethanesulfonyl)imide to the filter membrane at a rate of 3 μL / s, incubate with shaking at 25°C for 2 minutes to precipitate the extractant and reduce the number of washing cycles, and centrifuge at 20000g to remove the waste liquid.
[0129] 5. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and centrifuge at 20000g to clean the filter membrane. Repeat this cleaning operation 4 times.
[0130] 6. Add 100 μL of 20 mM ABC buffer solution to the filter membrane and add 10 μg of trypsin (protein to hair protein mass ratio 1:7.5). Incubate at 37°C for 4.5 hours. After centrifugation at 20000g, the filtrate is the peptide solution.
[0131] 7. The peptide solution was analyzed using liquid chromatography-mass spectrometry:
[0132] The liquid chromatography and mass spectrometry conditions were the same as in Example 1.
[0133] As a result, the recovery rate of sample 008 was 30.3%, and 1649 peptides and 231 proteins were identified. The main reason for the low recovery rate and lower identification coverage than in Examples 1-5 was that the sample was not heated and soaked after the extraction reagent was added, the hair shaft was not softened enough, and the internal disulfide bonds were not fully destroyed, which reduced the solubility of the protein, resulting in a low extraction recovery rate and insufficient protein content, leading to low identification coverage.
[0134] Example 9
[0135] 1. Preparation of extraction reagent: Mix 6M guanidine hydrochloride and 12% (w / w) solution of 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) (aqueous solution) at a volume ratio of 1:6, and add tris(2-carboxyethyl)phosphine (TCEP) to make the final concentration 200mM;
[0136] 2. Wash a 1 cm long hair shaft (number 009, human hair) with an 80% methanol solution for 5 minutes using sonication to remove the washing solution. Cut the hair shaft into pieces about 1 mm in size and add 100 μL of extraction reagent (0.6 mg hair shaft / mL extraction reagent). Add 100 μL of 0.5 M sodium hydroxide solution to adjust the pH to 7.5. Soak the hair shaft at 50°C with shaking for 1 hour to soften it. Use contact sonication for 20 minutes until there are no visible particles in the hair shaft suspension. Centrifuge at 20,000 g for 5 minutes and collect the supernatant, which is the hair shaft protein extraction solution (hair shaft protein solution).
[0137] 3. Add 100 μL of 200 mM TCEP (the volume ratio of hair dry protein solution to TCEP solution is 1:0.5) to the supernatant, denature and reduce at 95°C for 3 minutes, place at room temperature, transfer to a filter membrane with a molecular weight cutoff of 10 kDa, centrifuge to remove waste liquid, add 100 μL of 50 mM iodoacetamide solution to the filter membrane, incubate at room temperature in the dark for 30 minutes, centrifuge at 20000g to remove reaction solution, add 100 μL of 50 mM phosphate (PBS) buffer solution to the filter membrane, and centrifuge at 20000g to wash the filter membrane;
[0138] 4. Add 100 μL of 50 mM PBS buffer solution to the filter membrane and centrifuge at 20,000 g to wash the filter membrane. Repeat this washing operation 20 times.
[0139] 5. Add 100 μL of 50 mM PBS buffer solution to the filter membrane, and add 5 μg of Lys-C enzyme (protease to hair protein mass ratio 1:5). Incubate at 37°C for 1 hour. After centrifugation at 20000g, the filtrate is the peptide solution.
[0140] 6. The peptide solution was analyzed using liquid chromatography-mass spectrometry (LC-MS):
[0141] The chromatographic system was an EASY-nano LCTM1200;
[0142] The chromatographic column is a C18 reversed-phase capillary column with an inner diameter of 150 micrometers and a length of 20 centimeters;
[0143] The mobile phase consists of mobile phase A and mobile phase B in a certain proportion for gradient elution;
[0144] Mobile phase A is 2% acetonitrile (containing 0.1% formic acid by volume);
[0145] Mobile phase B is 80% acetonitrile (containing 0.1% formic acid by volume);
[0146] The flow rate of the mobile phase is 600 nanoliters per minute;
[0147] The gradient (V / V) is as follows: 0-28 min, 5%-16%B; 28-58 min, 16%-30%B; 58-77 min, 30%-45%B; 77-78 min, 45%-95%B; 78-85 min, 95%B.
[0148] Mass spectrometry conditions are:
[0149] The mass spectrometry system was an Orbitrap Fusion Lumos.
[0150] Data acquisition was performed using a non-data-dependent mode. Full MS scan range was 350-1322 m / z, resolution was 60000, automatic gain control (AGC) was 4e5, and maximum injection time (IT) was 50 ms. MSMS parameters were: resolution 30000, AGC 5e5, IT 50 ms, separation window 37 m / z, fragmentation mode High-energy collisional dissociation (HCD), and normalized collision energy 30%. The results showed that sample 009 had a recovery rate of 32.8%, identifying 682 peptides and 83 proteins. The low recovery rate and lower identification coverage compared to Examples 1-5 were mainly due to the absence of an extraction reagent precipitant added to the filter membrane. Even with increased washing cycles, the extraction reagent could not be completely removed, affecting enzyme activity and reducing enzymatic digestion efficiency. Furthermore, the extraction reagent interfered with ionization after entering the mass spectrometer, resulting in low identification coverage. Additionally, the extraction reagent entering the capillary column was difficult to elute, easily causing nozzle clogging and intermittent spraying (see...). Figure 9 This is also one of the reasons for low identification coverage. In addition, the increased number of washing cycles increases sample pretreatment time by several hours, which is not conducive to high-throughput sample analysis.
Claims
1. A rapid pretreatment method for hair shaft proteome samples, characterized in that: 1) The hair shaft was ultrasonically washed with methanol / water solution to remove the washing solution and obtain the hair shaft sample; 2) The extraction reagent is a mixed solution of guanidine hydrochloride, 1-dodecyl-3-methylimidazole chloride (C12 Im-Cl), and tris(2-carboxyethyl)phosphine (TCEP). Sodium hydroxide solution is added to the extraction reagent to adjust the pH to 7-8. The sample is then added to the hair sample, heated, shaken, and soaked to soften the hair. The hair is then broken up by ultrasonication. 3) Centrifuge the broken hair shaft suspension and take the supernatant as the hair shaft protein extraction solution. Add tris(2-carboxyethyl)phosphine (TCEP) solution to the hair shaft protein extraction solution to further denature and reduce the protein. 4) Transfer the solution to a filter membrane with a molecular weight cutoff of 2-20 kDa (preferably 2-15 kDa, more preferably 10-12 kDa), centrifuge to remove the waste liquid, add an alkylating agent to the filter membrane to carry out the alkylation reaction, centrifuge to remove the reaction liquid, and add a cleaning solution to the filter membrane to clean it. 5) Add the extraction reagent precipitant to the filter membrane at a rate of 1-10 (preferably 2-8, more preferably 5-6) μL / s, and incubate with shaking at 20℃-35℃ (preferably 23℃-32℃, more preferably 24℃-25℃) for 1-10 minutes (preferably 2-8 minutes, more preferably 2-3 minutes), and centrifuge to remove the waste liquid; 6) Add cleaning solution to the filter membrane, centrifuge to clean the filter membrane, and repeat this operation 0-20 times (preferably 2-15 times, more preferably 4-5 times); 7) Add washing solution and protease to the filter membrane, enzymatically hydrolyze at 36℃-38℃, and the filtrate after centrifugation is the peptide solution.
2. The pretreatment method according to claim 1, characterized in that: The extraction reagent is obtained by mixing guanidine hydrochloride solution and 1-dodecyl-3-methylimidazole chloride (C12 Im-Cl) solution and then adding TCEP; the molar concentration of the guanidine hydrochloride solution is 4-8M (preferably 5-7M, more preferably 6-6.5M), the mass concentration of the C12 Im-Cl solution is 2%-20% (preferably 5%-16%, more preferably 10%-12%), the mixing ratio of the guanidine hydrochloride solution and the C12 Im-Cl solution is 1:2-8 (preferably 1:3-6, more preferably 1:4-5) by volume, and the final concentration of TCEP contained in the extraction reagent is 10-900mM (preferably 50-500mM, more preferably 200-250mM).
3. The pretreatment method according to claim 1, characterized in that: The extracting reagent precipitant is a salt that can form a low-solubility precipitate with C12 Im-Cl, including at least one or more of bis(trifluoromethane)sulfonylimide salt, chlorine tetroxide, hexafluorophosphate, and bis(trifluoromethanesulfonylimide salt). The solution mass concentration is 10%-100% (preferably 20%-80%, more preferably 40%-50%), and the added volume is 30-150 μL (preferably 40-120 μL, more preferably 60-80 μL).
4. The pretreatment method according to claim 1, characterized in that: The hair shaft is 0.5-10 cm in length (preferably 1-8 cm, more preferably 2-4 cm). 2-10 ml of a methanol / water solution with a volume fraction of 40%-80% (preferably 50%-70%, more preferably 60%-65%) is added for washing. After washing, an extraction reagent is added to the hair shaft at a ratio of 0.2-10 (preferably 0.5-5, more preferably 2-4) mg hair shaft / ml extraction reagent. The molar mass of the sodium hydroxide is 0.1-1M (preferably 0.2-0.8M, more preferably 0.4-0.6M). After adding the extraction reagent, the hair shaft is shaken and soaked at 30℃-95℃ (preferably 35℃-80℃, more preferably 40℃-60℃) for 0.5-3 hours (preferably 1-3 hours, more preferably 2-2.5 hours), followed by ultrasonication for 5-90 minutes (preferably 10-60 minutes, more preferably 15-30 minutes).
5. The pretreatment method according to claim 1 or 4, characterized in that: Add TCEP solution with a molar concentration of 10-900 mM (preferably 50-500 mM, more preferably 300-400 mM) to the hair dry protein solution at a volume ratio of 1:0.3-2 (preferably 1:0.5-1.5, more preferably 1:1-1.2), and heat in a water bath at 40℃-95℃ (preferably 50℃-95℃, more preferably 80℃-90℃) for denaturation and reduction for 2-15 minutes (preferably 4-12 minutes, more preferably 6-8 minutes).
6. The pretreatment method according to claim 1 or 4, characterized in that: The alkylating agent is at least one or more of iodoacetamide or chloroacetamide. The volume of 30-80 mM alkylating agent solution added to the filter membrane is 50-150 μL. The alkylation reaction time is 10-50 minutes (preferably 20-40 minutes, more preferably 15-20 minutes).
7. The pretreatment method according to claim 1 or 4, characterized in that: The cleaning solution is at least one or more of ammonium bicarbonate (ABC) buffer solution or phosphate buffer solution, with a molar concentration of 10-100 mM (preferably 20-80 mM, more preferably 50 mM), a pH of 7-9 (preferably 7.5-8), and a volume of 50-150 μL (preferably 80-120 μL, more preferably 100 μL) added to the filter membrane.
8. The pretreatment method according to claim 1, characterized in that: The protease is at least one or more of trypsin, Lys-C enzyme, Glu-C enzyme, Arg-C enzyme, and Asp-N enzyme. The protease is added to the filter membrane at a ratio of protease to hair shaft protein of 1:1-50 (preferably 1:5-40, more preferably 1:10-20) and enzymatically hydrolyzed at 36℃-38℃ for 0.5-6 hours (preferably 1-5 hours, more preferably 3-4 hours).
9. The pretreatment method according to claim 1, characterized in that: The obtained peptide solution is either cryopreserved or directly analyzed using liquid chromatography-mass spectrometry (LC-MS), wherein the detection conditions for LC-MS include: The liquid chromatography conditions are as follows: The chromatographic column is a reversed-phase capillary column with an inner diameter of 50 micrometers to 1 millimeter and a length of 5 to 50 centimeters; The mobile phase consists of mobile phase A and mobile phase B in a certain proportion for gradient elution; Mobile phase A is 0%-2% acetonitrile (containing 0.01%-0.5% formic acid, acetic acid, and trifluoroacetic acid, or at least two or more of these by volume); Mobile phase B is 80%-100% acetonitrile (containing 0.01%-0.5% formic acid, acetic acid, and trifluoroacetic acid, or at least two of these by volume); The flow rate of the mobile phase is 100-1000 nanoliters per minute; The mass spectrometry conditions were as follows: at least one of the following was used: electrospray ionization source, atmospheric pressure chemical ionization source or matrix-assisted laser desorption / ionization source, positive ion mode, and at least one of the following acquisition methods: data-dependent or data-independent.
10. The pretreatment method according to any one of claims 1-9, characterized in that: This preprocessing method can be applied to one or more fields, including individual identification in forensic medicine, disease pathogenesis research, diagnostic markers, and therapeutic target discovery in the medical field, species identification, species origin, and population genetic structure analysis in animal taxonomy, and archaeology.