High-throughput extraction method and application of hair stem sample protein
Through pressure cycling sample preparation technology and enzymatic treatment, the problem of high-throughput extraction of hair shaft proteins is solved, and the interference of lipids and impurities is effectively removed, and the analysis coverage depth and detection sensitivity of hair shaft proteome are improved.
Patent Information
- Application Number
- CN202410082832.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-19
- Publication Date
- 2025-07-22
AI Technical Summary
The prior art is difficult to extract hair shaft proteins for mass spectrometry for high throughput, and there are problems such as large sample usage, long enzymatic decomposition time, and small number of protein detection, which cannot meet the requirements of trace and rapid processing.
Pressure cycle sample preparation technology (PCT) was used to extract proteins from ex vivo hair shafts, and combined with a mixture of lysate and reducing agent for hair shaft protein extraction and enzymatic treatment, including multiple normal pressure and ultra-high hydraulic cycles to remove interference from lipids and impurities, and enzymatically lysC and trypsin were used for enzymatic decomposition.
It has achieved efficient removal of lipid and impurities interference from hair dry samples, and can detect hair dry proteome with high sensitivity, improve the analysis coverage depth, simplify the processing flow, and is suitable for high-throughput pretreatment of 16 hair dry samples.
Smart Images
Figure BDA0004673705440000101 
Figure HDA0004673705530000011
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological detection, and particularly relates to a high-throughput extraction method and application of hair shaft sample proteins. Background Art
[0002] Hair shafts are rich in proteins, with more than 90% of their components being composed of proteins. The types and sequence information of hair shaft proteins contain abundant genetic information. Each person sheds about one hair every 10 to 15 minutes, resulting in hair shafts being present at many crime scenes and being a very common biological evidence. By detecting the hair shaft proteins at the scene, the suspect can be linked to the victim or the suspect to the crime scene. Therefore, hair shaft protein testing has broad forensic application potential.
[0003] Hair shafts are mainly composed of keratin and hydrophobic proteins of keratin-associated proteins (KAPs), and the proteins are cross-linked by a large number of disulfide bonds. They have good stability and are still difficult to dissolve even in strong denaturants, which is not conducive to protease digestion and poses challenges to hair shaft proteome analysis based on liquid chromatography-mass spectrometry systems.
[0004] Currently, the hair shaft sample dissolution system still follows the system for analyzing samples such as cells and body fluids. One of the most commonly used schemes is the Shindai method (Preparation and properties of protein films and particles from chicken feather, 2008; Vol.8), which mainly uses 20 mM Tris-HCl (pH 8.5), 2.6 M thiourea, 5 M urea, and 5% (v / v) 2-mercaptoethanol (2-ME) as extraction reagents and incubates at 50 °C for 24 h for protein extraction. Another optimized extraction method for human hair proteins (Glendon J. Parker Optimal Processing for Proteomic Genotyping of Single Human Hairs, Forensic Science International: Genetics, (2020) 47(102314):1-10) uses 2% SD + 50 mM ABC + 100 mM dithiothreitol (DTT) as extraction reagents. First, it reduces with 100 mM DDT at room temperature for 6 hours, alkylates with 200 mM iodoacetamide for 45 minutes, and digests with 1:50 (trypsin: protein) for 6 hours, detecting an average of 2700 specific peptide segments. Such methods have disadvantages such as large sample consumption, long enzymatic digestion time, few protein detections, and inability to perform high-throughput detection, and cannot meet the requirements of microscale and rapid processing.
[0005] In addition, at present, the hair shaft mainly focuses on qualitative analysis in the field of forensic identification, lacking quantitative data. The proteomic research on the hair shaft still remains at the stage of only being able to identify medium- and high-abundance proteins, lacking a comprehensive description of the hair shaft proteome and research on its application in forensic identification. Therefore, there is great potential for exploring the hair shaft proteome extraction technology. Summary of the Invention
[0006] The main problem to be solved by the present invention is how to extract hair shaft proteins for mass spectrometry identification in a high-throughput manner.
[0007] To solve the above problems, the present invention provides a method for preparing hair shaft protein peptides for mass spectrometry analysis.
[0008] The method for preparing hair shaft protein peptides for mass spectrometry analysis provided by the present invention includes the following steps:
[0009] Using Pressure Cycling Technology (PCT) to extract proteins from ex vivo hair shafts to obtain hair shaft proteins; performing enzymatic digestion on the hair shaft proteins to obtain hair shaft protein peptides for mass spectrometry analysis.
[0010] In the above method, when the Pressure Cycling Technology is used for extraction, the number of cycles is 90 - 280 times.
[0011] In the above method, the number of cycles of the Pressure Cycling Technology is the most critical parameter, and other parameters can be set according to the specific PCT pressure cycling instrument equipment used according to the equipment parameters.
[0012] In a specific embodiment, the Pressure Cycling Technology uses the following extraction parameters for extraction: 45 kpsi, 50 s HP, 10 s AP; 90 - 280 cycles, and the working temperature can be 70 °C.
[0013] In the above method, the extraction includes using a mixed solution containing a lysis solution and a reducing agent as an extraction reagent to extract proteins from the ex vivo hair shafts to obtain a first treatment solution; performing enzymatic digestion of the hair shaft proteins on the first treatment solution to obtain a second treatment solution; performing desalting, concentration, and reconstitution on the second treatment solution to obtain hair shaft protein peptides for mass spectrometry analysis.
[0014] In the above method, the first treatment solution is prepared according to the following steps:
[0015] 1) Dissolve the ex vivo hair shaft sample described above with the extraction reagent;
[0016] 2) Extraction by pressure cycling sample preparation technique: The extraction parameters of hair shaft protein by the pressure cycling sample preparation technique are: 45 kpsi, 50 s HP, 10 s AP; 90 - 280 cycles, 70 °C.
[0017] 3) Add iodoacetamide to terminate the reaction to obtain the first treatment solution.
[0018] In the above method, the reducing agent is TCEP.
[0019] In the above method, in the mixed solution containing the lysis solution and the reducing agent, the concentration of the reducing agent is 10 mM - 50 mM.
[0020] In the above method, the obtaining of the ex vivo hair shaft sample further includes a step of defatting the hair shaft. The defatting step uses an aqueous ethanol solution and performs ultrasonic bath for 20 s - 30 min.
[0021] In the above method, the enzymatic digestion treatment is performed using lysC and trypsin for enzymatic digestion.
[0022] The application of the above-mentioned preparation method in the high-throughput extraction and analysis of hair shaft protein also belongs to the scope of protection of the present invention.
[0023] The present invention uses the pressure cycling sample preparation technique to extract protein from ex vivo hair shafts to obtain hair shaft protein; the enzymatic digestion treatment of hair shaft protein is performed to obtain hair shaft protein peptides for mass spectrometry analysis, and has the following advantages: 1) It can effectively remove the interference of lipids and impurities on the hair shaft sample and reduce the interference of pollutants on subsequent proteomic analysis; 2) The pressure cycling sample preparation technique is adopted, and the hair shaft protein is extracted and lysed by rapidly cycling the pressure between normal pressure and ultra-high hydraulic pressure multiple times; 3) This method can extract trace hair shaft samples with a length of 2 cm, which is beneficial to the high-sensitivity detection of hair shaft protein; 4) This method realizes efficient extraction and protein enzymatic digestion, and improves the analysis coverage depth of the hair shaft proteome; 5) The method steps are simple, and the protein group sample pretreatment of 16 hair shaft samples can be carried out simultaneously. Description of the Drawings
[0024] Figure 1 It is the mass spectrometry detection result of Example 6. Detailed Embodiments
[0025] The present invention will be further described in detail below in conjunction with the specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.
[0026] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
[0027] The composition of the lysis buffer in the following examples is as follows: 6M urea and 2M thiourea, and the rest is water.
[0028] The preparation method of the 200mM TCEP solution in the following examples is as follows: 0.115g of TCEP solid (Tris(2-carboxyethyl)phosphine) is dissolved in 2ml of 25mM Tris buffer with a pH of 8.
[0029] The preparation method of the 800mM IAA solution in the following examples is as follows: 0.1480g of iodoacetamide (abbreviated as IAA) is dissolved in 1mL of 25mM Tris buffer with a pH of 8. The 25mM Tris buffer is prepared by dissolving 3.0275g of Tris in 800mL of water, adjusting the pH to 8.0 with concentrated hydrochloric acid, and finally making up to 1L.
[0030] In the following examples, lysC is lysine-specific protease, which is a product with the catalog number VA1170 of Promega Corporation.
[0031] In the following examples, trypsin is trypsin, which is a product with the catalog number VA9000 of Promega Corporation.
[0032] Example 1. Establishment of a high-throughput protein extraction method for hair shaft samples
[0033] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method:
[0034] 1. Pretreatment of hair shaft samples:
[0035] Take a 2-cm-long human hair shaft in a 1.5-mL centrifuge tube, add 1mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), shake it with a vortex mixer for 1 min and then discard the solution. Add another 1mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), and perform ultrasonic bath for 20 s to remove the grease and impurities on the surface of the hair shaft. Cut the hair shaft into pieces with a cleaned ophthalmic scissors.
[0036] 2. Reduce, alkylate, and enzymatically digest the hair shaft protein using PCT technology:
[0037] A, PCT extraction: Dissolution of hair samples: Transfer the shredded hair shaft samples from step 1 to a PCT tube. Use a pipette to transfer out the above ethanol aqueous solution. After the sample is dried, add 30 μL of lysis buffer to the sample, and add 2.5 μL of 200 mM TCEP solution. The parameters of the PCT pressure cycling instrument (Barocycler 2320EXT, Pressure BioSciences Inc, SouthEaston, MA, USA) are set as follows: 45 kpsi, 50 s HP, 10 s AP; 90 cycles, 70 °C.
[0038] B, Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min, 800 rpm, 30 °C. Obtain the first treatment solution.
[0039] C, Enzymolysis: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all the first treatment solutions, and use Tris buffer to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C, add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction, and obtain the second treatment solution.
[0040] 4, C18 desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, 1 time; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, 1 time; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, 1 time; ④ 160 μL of the second treatment solution (80 μl, repeat 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeat 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, 1 time.
[0041] 5, Centrifuge and concentrate for 30 min. Redissolve with 5 μL of MS buffer, measure the concentration with a nanodrop, and the peptide recovery is 0.55 μg.
[0042] Example 2
[0043] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method.
[0044] 1. Pretreatment of hair samples: Take a 2-cm human hair sample and place it in a 1.5-mL centrifuge tube. Add 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1). After vortexing for 1 min, discard the solution. Then add another 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), and perform ultrasonic bath for 10 min to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a clean ophthalmic scissors.
[0045] 2. Use PCT technology to reduce, alkylate, and enzymatically digest hair shaft proteins:
[0046] A. PCT extraction: Dissolution of hair samples: Transfer the shredded hair shaft sample in Step 1 to a PCT tube. Use a pipette to transfer out the above ethanol aqueous solution. After the sample is dried, add 30 μL of lysis buffer and 5 μL of 100 mM TCEP to the sample. The parameters of the PCT pressure cycling instrument are set as follows: 45 kpsi, 50 s HP, 10 s AP; 90 cycles, 70 °C.
[0047] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min, 800 rpm, 30 °C. Obtain the first treatment solution.
[0048] C. Enzymatic digestion: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction and obtain the second treatment solution.
[0049] 3. C18 desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of the sample (80 μl, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeat 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0050] 4. Centrifugally concentrate for 30 min. Resuspend with 5 μL of MS buffer and measure the concentration using a nanodrop. The recovered amount of peptides is 4.15 μg.
[0051] Perform proteomic mass spectrometry analysis (ThermoFisher QE mass spectrometry) based on a liquid chromatography - mass spectrometry system. In the 60 - min DDA acquisition mode, use pFind software to identify proteins. A total of 4862 peptides were identified, corresponding to 1110 hair shaft proteins.
[0052] Example 3
[0053] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract hair shaft proteins according to the following method.
[0054] 1. Pretreatment of hair samples: Take a 2 - cm human hair sample in a 1.5 - mL centrifuge tube, add 1 mL of ethanol - aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), vortex for 1 min and then discard the solution. Add another 1 mL of ethanol - aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), and perform ultrasonic bath for 10 min to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a clean ophthalmic scissors.
[0055] 2. Reduce, alkylate, and enzymatically digest hair shaft proteins using the PCT technique:
[0056] A. PCT extraction: Dissolution of hair samples: Transfer the shredded hair shaft sample from step 1 to a PCT tube, use a pipette to transfer out the above - mentioned ethanol - aqueous solution. After the sample is dried, add 30 μL of lysis buffer and 2.5 μL of 100 mM TCEP to the sample. The parameters of the PCT pressure cycling instrument are set as: 45 kpsi, 50 s HP, 10 s AP,; 180 cycles, 70 °C.
[0057] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min, 800 rpm, 30 °C, to obtain the first treatment solution.
[0058] C. Enzymatic digestion: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL, and use Tris - HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction and obtain the second treatment solution.
[0059] 3. C18 Desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of the sample (80 μl, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0060] 4. Centrifugal concentration for 30 min. Reconstitute with 5 μL of MS buffer and measure the concentration using a nanodrop. The recovered amount of peptides is 1.45 μg.
[0061] Perform proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on a liquid chromatography-mass spectrometry system. In the 60-min DDA acquisition mode, use the pFind software to identify proteins. A total of 5052 peptide segments were identified, corresponding to 1321 hair shaft proteins.
[0062] Example 4
[0063] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract hair shaft proteins according to the following method.
[0064] 1. Pretreatment of hair samples: Take a 2-cm human hair sample in a 1.5-mL centrifuge tube, add 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), shake with a vortex mixer for 1 min and then discard the solution. Add another 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), and perform ultrasonic bath for 20 s to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a clean ophthalmic scissors.
[0065] 2. Reduce, alkylate, and enzymatically digest hair shaft proteins using the PCT technique:
[0066] A. PCT extraction: Dissolution of hair samples: Transfer the cut hair shaft sample in step 1 to a PCT tube, use a pipette to transfer out the above ethanol aqueous solution. After the sample is dried, add 30 μL of lysis buffer and 5 μL of 100 mM TCEP to the sample. The parameter settings of the PCT pressure cycling instrument are: 45 kpsi, 50 s HP, 10 s AP, 180 cycles, 70 °C.
[0067] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min at 800 rpm and 30 °C to obtain the first treatment solution.
[0068] C, Enzymatic digestion: Add 2.5 μL of lysC aqueous solution (original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction and obtain the second treatment solution.
[0069] 3. C18 desalting: ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of sample (80 μL, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0070] 4. Centrifugal concentration for 30 min. Reconstitute with 5 μL of MS buffer and measure the concentration with a nanodrop. The peptide recovery is 0.65 μg.
[0071] Example 5
[0072] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method.
[0073] 1. Pretreatment of hair sample: Take a 2 cm human hair sample in a 1.5 mL centrifuge tube, add 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), shake with a vortex mixer for 1 min and then discard the solution. Add another 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), and perform water bath sonication for 20 s to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a clean ophthalmic scissors.
[0074] 2. Reduce, alkylate, and enzymatically digest the hair shaft protein using PCT technology:
[0075] A, PCT extraction: Dissolution of hair samples: Transfer the shredded hair shaft samples in Step 1 to a PCT tube. Use a pipette to transfer out the above ethanol aqueous solution. After the sample is dried, add 30 μL of lysis buffer to the sample and add 5 μL of 100 mM TCEP. The parameters of the PCT pressure cycling instrument are set as: 45 kpsi, 50 s HP, 10 s AP, 270 cycles, 70 °C.
[0076] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction. Shake it in the dark for 30 min at 800 rpm and 30 °C to obtain the first treatment solution.
[0077] C. Enzymolysis: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all the first treatment solutions. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction to obtain the second treatment solution.
[0078] 3. C18 desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of the sample (80 μL, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0079] 4. Centrifuge and concentrate for 30 min. Re-dissolve with 5 μL of MS buffer and measure the concentration with a nanodrop. The recovered amount of peptides is 1 μg. Perform proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on the liquid chromatography-mass spectrometry system. Use the pFind software to identify proteins in the 60-min DDA acquisition mode. A total of 7,817 peptides were identified, corresponding to 1,768 hair shaft proteins.
[0080] Example 6
[0081] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method.
[0082] 1. Pretreatment of hair samples: Take a 2-cm human hair sample and place it in a 1.5-mL centrifuge tube. Add 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1). After vortexing for 1 min, discard the solution. Then add another 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), and perform ultrasonic bath for 30 s to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a cleaned ophthalmic scissors.
[0083] 2. Use PCT technology to reduce, alkylate, and enzymatically digest hair shaft proteins:
[0084] A. PCT extraction: Dissolution of hair samples: Transfer the cut hair shaft samples in step 1 to a PCT tube. Use a pipette to transfer out the above ethanol aqueous solution. After the sample is dried, add 30 μL of lysis buffer and 2.5 μL of 200 mM TCEP to the sample. The parameters of the PCT pressure cycling instrument are set as follows: 45 kpsi, 50 s HP, 10 s AP, 120 cycles, 70 °C.
[0085] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min at 800 rpm and 30 °C to obtain the first treatment solution.
[0086] C. Enzymatic digestion: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction to obtain the second treatment solution.
[0087] 3. C18 desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of the sample (80 μL, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0088] 4. Centrifuge and concentrate for 30 min. Resuspend in 5 μL of MS buffer and measure the concentration using a nanodrop. The recovered amount of peptides is 0.85 μg. Perform proteomic mass spectrometry analysis (ThermoFisher QE mass spectrometry) based on a liquid chromatography-mass spectrometry system. In the 60-min DDA acquisition mode, use the pFind software to identify proteins. A total of 10,171 peptides were identified, corresponding to 2,241 hair shaft proteins. The identification results are as Figure 1 shown.
[0089] Example 7
[0090] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method.
[0091] 1. Pretreatment of hair sample: Take a 2-cm human hair sample in a 1.5-mL centrifuge tube, add 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), shake for 1 min with a vortex mixer and then discard the solution. Add another 1 mL of ethanol aqueous solution (prepared by mixing ethanol and water in a volume ratio of 1:1), and perform water bath sonication for 30 min to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into small pieces with a clean ophthalmic scissors.
[0092] 2. Reduce, alkylate, and digest the hair shaft protein using the PCT technique:
[0093] A. PCT extraction: Dissolution of hair sample: Transfer the shredded hair shaft sample in step 1 to a PCT tube, pipette out the above ethanol aqueous solution, and after the sample is dried, add 30 μL of lysis buffer and 1.5 μL of 1 M TCEP to the sample. Set the parameters of the PCT pressure cycling instrument as: 45 kpsi, 50 s HP, 10 s AP, 200 cycles, 70 °C.
[0094] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min at 800 rpm and 30 °C to obtain the first treatment solution.
[0095] C. Digestion: Add 2.5 μL of lysC (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the digestion reaction and obtain the second treatment solution.
[0096] 3. C18 Desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of the sample (80 μL, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0097] 4. Centrifugal concentration for 30 min. Reconstitute with 5 μL of MS buffer, measure the concentration with a nanodrop, and the peptide recovery is 3.4 μg. Perform proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on the liquid chromatography-mass spectrometry system. In the 60-min DDA acquisition mode, use the pFind software to identify proteins. A total of 2630 peptide segments were identified, corresponding to 430 hair shaft proteins.
[0098] Example 8
[0099] Take 1 human hair, remove the hair follicle, and take the hair shaft. Extract the hair shaft protein according to the following method.
[0100] 1. Pretreatment of hair sample: Take a 2-cm human hair sample in a 1.5-mL centrifuge tube, add 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), shake with a vortex mixer for 1 min and then discard the solution. Add another 1 mL of ethanol aqueous solution (mixed by ethanol and water with a volume ratio of 1:1), and perform ultrasonic bath for 30 s to remove the grease and impurities on the hair shaft surface. Cut the hair shaft into pieces with a clean ophthalmic scissors.
[0101] 2. Reduce, alkylate, and enzymatically digest the hair shaft protein using the PCT technique:
[0102] A. PCT extraction: Dissolution of hair sample: Transfer the shredded hair shaft sample in step 1 to a PCT tube, transfer out the above ethanol aqueous solution with a pipette. After the sample is dried, add 30 μL of lysis buffer to the sample, and add 1.5 μL of 1 M TCEP. The parameters of the PCT pressure cycling instrument are set as: 45 kpsi, 50 s HP, 10 s AP, 280 cycles, 70 °C.
[0103] B. Add 2.5 μL of 800 mM IAA solution to terminate the reaction, shake in the dark for 30 min, 800 rpm, 30 °C, to obtain the first treatment solution.
[0104] C, Enzymatic digestion: Add 2.5 μL of lysC aqueous solution (the original concentration of lysC is 0.25 μg / μL) and 10 μL of trypsin aqueous solution (the original concentration of trypsin is 0.25 μg / μL) to all of the first treatment solution. The concentrations of both lysC and trypsin are 0.25 μg / μL. Use Tris-HCl to make up the volume to 150 μL. Set the PCT parameters: 20 kpsi, 50 s HP, 10 s AP, 120 cycles, 30 °C. Add 15 μL of 10% TFA solution to terminate the enzymatic digestion reaction and obtain the second treatment solution.
[0105] 3. C18 desalting: Desalt the second treatment solution. ① 60 μL of MeOH, centrifuge at 1200 rpm for 1 min, once; ② 60 μL of 80% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ③ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1200 rpm for 1 min, once; ④ 160 μL of sample (80 μL, repeated 2 times, centrifuge at 1400 rpm); ⑤ 60 μL of 2% ACN, 0.1% TFA, centrifuge at 1450 rpm for 1 min, repeated 3 times; ⑥ 60 μL of 40% ACN (MS grade), 0.1% TFA, centrifuge at 1450 rpm for 1 min, once.
[0106] 4. Centrifugal concentration for 30 min. Reconstitute with 5 μL of MS buffer and measure the concentration using a nanodrop. The peptide recovery is 1.45 μg. Perform proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on a liquid chromatography-mass spectrometry system. In the 60-min DDA acquisition mode, use the pFind software to identify proteins. A total of 1966 peptide segments were identified, corresponding to 429 hair shaft proteins.
[0107] Example 9
[0108] The sample pretreatment process is the same as that in Example 6, and the peptide recovery is 1.6 μg. Perform proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on a liquid chromatography-mass spectrometry system. In the 60-min DDA acquisition mode, use the pFind software to identify proteins. A total of 5800 peptide segments were identified, corresponding to 1650 hair shaft proteins.
[0109] Example 10
[0110] During the sample pretreatment process, the PCT extraction parameter - the number of cycles was changed to 180, and other processes were the same as in Example 6. The peptide recovery was 1.3 μg. Proteomic mass spectrometry (ThermoFisher QE mass spectrometry) analysis based on a liquid chromatography-mass spectrometry system was performed. In the 60-minute DDA acquisition mode, the pFind software was used to identify proteins. A total of 7,745 peptides were identified, corresponding to 1,782 hair shaft proteins.
[0111] Table 1. Protein extraction information under different experimental conditions in each embodiment of the present invention
[0112]
[0113] Note: In Table 1, "\ " indicates that the peptide recovery was low and no measured data was available.
[0114] Table 1 shows the extraction of protein peptides in each embodiment under different experimental conditions. The above results indicate that: the method established by the present invention has simple steps, can effectively remove the interference of lipids and impurities on the hair shaft sample, and can extract proteins in the hair shaft with a high recovery rate, efficiently perform protein enzymatic hydrolysis, and achieve high-sensitivity coverage depth analysis of the hair shaft proteome.
[0115] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.
Claims
1. A method for preparing hair shaft protein peptides for mass spectrometry analysis, characterized in that, Comprising the following steps: Extracting protein from ex vivo hair shafts using pressure cycling sample preparation technology to obtain hair shaft protein; Performing enzymatic digestion on the hair shaft protein to obtain hair shaft protein peptides for mass spectrometry analysis.
2. The method according to claim 1, wherein In the pressure cycling sample preparation technology, the number of cycles is 90 - 280 times.
3. The method according to claim 1, wherein The pressure cycling sample preparation technology is used for extraction with the following extraction parameters: 45 kpsi, 50 s HP, 10 s AP; the number of cycles is 90 - 280 times, and the working temperature is 70°C.
4. The method according to any one of claims 1 to 3, characterized in that The extraction includes using a mixed solution containing a lysis solution and a reducing agent as an extraction reagent to extract protein from the ex vivo hair shafts to obtain a first treatment solution; performing enzymatic digestion of the hair shaft protein on the first treatment solution to obtain a second treatment solution; Performing desalting, concentration, and reconstitution on the second treatment solution to obtain hair shaft protein peptides for mass spectrometry analysis.
5. The method according to claim 4, wherein The first treatment solution is prepared by a method comprising the following steps: 1) Dissolving the ex vivo hair shaft sample described in claim 1 with the extraction reagent; 2) Extracting using pressure cycling sample preparation technology: The extraction parameters for the hair shaft protein using the pressure cycling sample preparation technology are: 45 kpsi, 50 s HP, 10 s AP; 90 - 280 cycles, 70°C; 3) Adding iodoacetamide to terminate the reaction to obtain the first treatment solution.
6. The method according to claim 4 or 5, characterized in that, The reducing agent is TCEP.
7. According to the method according to any one of claims 4-6, characterized in that In the mixed solution containing the lysis solution and the reducing agent, the concentration of the reducing agent is 10 mM - 50 mM.
8. The method according to any one of claims 1-7, characterized in that, The obtaining of the ex vivo hair shaft sample further includes a step of defatting the hair shaft, and the defatting step uses an aqueous ethanol solution and performs water bath ultrasound for 20 s - 30 min.
9. The method according to any one of claims 1-8, characterized in that The enzymatic digestion treatment is performed using lysC and trypsin for enzymatic digestion.
10. Use of the method according to any one of claims 1 - 9 in high - throughput extraction and analysis of hair shaft protein.