Enzymatic digestion method for gently reducing agent to digest tissue samples and its mass spectrometry detection method

Abstract

The present invention discloses an enzymatic digestion method for gently reducing agent to digest tissue samples and its mass spectrometry detection method. The present invention provides a lysis solution, which comprises: a detergent with a final concentration of 1-4% by mass fraction; the detergent is sodium deoxycholate and / or ammonium bicarbonate; 3-(2-carboxy)phosphine hydrochloride with a final concentration of 5-20 μM; 5-chloro-anthranilic acid with a final concentration of 20-60 mM; tris(hydroxymethyl)aminomethane-hydrochloride with a final concentration of 50-200 mM; phenylmethylsulfonyl fluoride with a final concentration of 0.1-2 mM; and water. After the tissue samples are lysed with the lysis solution of the present invention, tissue proteins and peptides can be obtained with a high yield.

Description

Technical Field

[0001] The present invention relates to an enzymatic hydrolysis method for gently reducing agent to hydrolyze tissue samples and a mass spectrometry detection method therefor. Background Art

[0002] For the acquisition of biopsy tissues or trace special tissue samples, the most commonly used method at present is microdissection. However, the scope of application of this method is limited, and it is mostly used for the cutting of frozen tissues, which is time-consuming, laborious and expensive. Moreover, for clinical samples, especially samples after minimally invasive surgery (such as biopsy tissues), the sample volume is small, the quantity is small, and the samples available for research and medical detection and evaluation are limited. Nowadays, the health and safety awareness of early detection, early screening and early diagnosis of human diseases has been significantly improved, and minimally invasive surgery has gradually become popular. Therefore, the technology and detection level of micro-sample segmentation (regional cutting) are very crucial and necessary.

[0003] Based on special tissue samples, especially paraffin-embedded tissue samples, it is difficult to extract tissue proteins cross-linked by disulfide bonds.

[0004] In addition, different from the large sample volume requirements of genomics, transcriptomics, metabolomics, etc., the upgrading and optimization of LC-MS / MS mass spectrometry can detect sample proteins with less cell and tissue amounts.

[0005] Therefore, by integrating the acquisition of micro-samples, protein extraction and mass spectrometry technology, the realization of protein detection for biopsy tissues or trace special tissue samples helps to accelerate the realization of early screening, early detection and early diagnosis of diseases. Summary of the Invention

[0006] The present invention provides an enzymatic hydrolysis method for gently reducing agent to hydrolyze tissue samples different from the prior art and a mass spectrometry detection method therefor. After the tissue samples are lysed by the lysate of the present invention, the yields of proteins and peptides are high, and the step of adding carbon powder to remove sodium deoxycholate is omitted during the extraction process, thereby simplifying the extraction steps, avoiding additional loss of proteins, and obtaining proteins in tissue samples with high yields. The lysis solution of the present invention is particularly suitable for samples with small amounts and small volumes (the volume is between 0.01 μL and 1 μL), Tissue samples or paraffin-embedded tissue samples.

[0007] The present invention provides a lysate, which comprises:

[0008] A detergent with a final concentration of 1-4% by mass fraction; the detergent is sodium deoxycholate (DOC) and / or ammonium bicarbonate (ABC);

[0009] 3-(2-carboxy)phosphine hydrochloride (TCEP) with a final concentration of 5-20 μM;

[0010] 5-chloro-anthranilic acid (CAA) with a final concentration of 20-60 mM;

[0011] Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) with a final concentration of 50 - 200 mM;

[0012] Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 0.1 - 2 mM;

[0013] and water.

[0014] In the present invention, the mass fraction of the detergent is the mass percentage of the mass of the detergent in the total mass of each raw material in the lysis solution. The molar concentration is the molar volume ratio of the molar amount of each component to the total volume of the lysis solution.

[0015] In the lysis solution, the detergent can be sodium deoxycholate.

[0016] In the lysis solution, the mass fraction of the detergent can be 1 - 3%, such as 1%, 2%, 3%.

[0017] In the lysis solution, the final concentration of 3-(2-carboxy)phosphine hydrochloride can be 10 - 20 μM, such as 10 μM, 20 μM.

[0018] In the lysis solution, the final concentration of 5-chloro-o-aminobenzoic acid can be 20 - 40 mM, such as 20 mM, 40 mM.

[0019] In the lysis solution, the final concentration of Tris(hydroxymethyl)aminomethane hydrochloride can be 50 - 100 mM, such as 50 mM, 100 mM.

[0020] In the lysis solution, the final concentration of phenylmethylsulfonyl fluoride can be 1 - 2 mM, such as 1 mM, 2 mM.

[0021] In the lysis solution, the pH value of the lysis solution can be 8.0 - 8.8.

[0022] The lysis solution can be a lysis solution for lysing tissue samples.

[0023] In the lysis solution, the tissue sample is preferably a biopsy tissue sample (e.g., with a volume ranging from 0.01 μL to 1 μL) or a paraffin-embedded tissue sample. The paraffin-embedded tissue sample is preferably human esophageal biopsy normal tissue paraffin-embedded tissue samples, human colorectal biopsy tissue paraffin-embedded tissue samples a paraffin-embedded tissue sample of human colorectal biopsy tissue, a paraffin-embedded tissue sample of human duodenal biopsy tissue, or a paraffin-embedded tissue sample of human gastric biopsy tissue. The paraffin-embedded sample of human colorectal biopsy tissue is preferably a paraffin-embedded tissue sample of in situ carcinoma. The paraffin-embedded tissue sample of human duodenal biopsy tissue is preferably a paraffin-embedded tissue sample of microinvasive carcinoma tissue. The paraffin-embedded tissue sample of human gastric biopsy tissue is preferably a paraffin-embedded tissue sample of poorly differentiated tissue.

[0024] In a preferred embodiment of the present invention, the lysis solution comprises a detergent with a mass fraction of 1-3%, 3-(2-carboxy)phosphine hydrochloride with a final concentration of 10 μM, 5-chloro-o-aminobenzoic acid with a final concentration of 20 mM, tris(hydroxymethyl)aminomethane-hydrochloride with a final concentration of 50 mM, phenylmethylsulfonyl fluoride with a final concentration of 2 mM, and water.

[0025] In a preferred embodiment of the present invention, the lysis solution comprises a detergent with a mass fraction of 1-3%, 3-(2-carboxy)phosphine hydrochloride with a final concentration of 20 μM, 5-chloro-o-aminobenzoic acid with a final concentration of 40 mM, tris(hydroxymethyl)aminomethane-hydrochloride with a final concentration of 100 mM, phenylmethylsulfonyl fluoride with a final concentration of 2 mM, and water.

[0026] In a preferred embodiment of the present invention, the lysis solution is composed of the detergent, the 3-(2-carboxy)phosphine hydrochloride, the 5-chloro-o-aminobenzoic acid, the tris(hydroxymethyl)aminomethane-hydrochloride, the phenylmethylsulfonyl fluoride, and the water.

[0027] The present invention also provides a method for preparing the above lysis solution, which comprises the following steps: mixing the components of the above lysis solution, heating, and cooling to room temperature to obtain the lysis solution.

[0028] Among them, the temperature of the heating can be 90-100 °C, such as 90 °C.

[0029] Among them, the time of the heating can be 5-60 min, such as 30 min.

[0030] Among them, the addition method of the detergent and phenylmethylsulfonyl fluoride in the raw materials is preferably added as needed.

[0031] The present invention also provides a kit for lysing tissue samples, which comprises the above lysis solution and at least one container for accommodating the above lysis solution.

[0032] The kit can be a kit for lysing tissue samples.

[0033] The present invention also provides an application of the above lysis solution in obtaining proteins and / or peptides in tissue samples.

[0034] Among them, the tissue samples are as described above.

[0035] The present invention also provides a method for obtaining proteins and / or peptides, which comprises the following steps:

[0036] Step 1: Lysing and denaturing the tissue sample in the above lysis solution to obtain mixture 1;

[0037] Step 2: Under the action of trypsin, the mixture obtained in Step 1 is enzymatically hydrolyzed to obtain Mixture 2;

[0038] Step 3: The Mixture 2 obtained in Step 2 is subjected to detergent removal treatment, extraction, and concentration to obtain Mixture 3;

[0039] Step 4: The mixture obtained in Step 3 is desalted to obtain proteins and / or peptides.

[0040] In Step 1, the tissue sample is as described above.

[0041] In Step 1, the temperature of the lysis and denaturation is preferably 90 - 100 °C, such as 90 °C. The time of the lysis and denaturation is preferably 5 - 60 min, such as 30 min.

[0042] In Step 2, the dosage of trypsin can be the conventional dosage in the art, preferably 40 - 60 μg / mL, such as 60 μg / mL.

[0043] In Step 2, the temperature of the enzymatic hydrolysis can be the conventional temperature in the art, preferably 30 - 40 °C, such as 37 °C.

[0044] In Step 2, the time of the enzymatic hydrolysis can be the conventional time in the art, preferably 12 - 24 h, such as 18 h.

[0045] In Step 2, the oscillation speed of the enzymatic hydrolysis can be the conventional oscillation speed in the art, preferably 300 - 1500 rpm, such as 1000 rpm.

[0046] In Step 3, the method of the detergent removal treatment preferably does not add a remover. The remover is preferably carbon powder.

[0047] In Step 3, the method of the detergent removal treatment preferably includes the following steps: under the presence of formic acid, oscillate for 1 - 5 min (such as 3 min), centrifuge at a speed of 8000 - 12500 rpm (such as 12000 rpm) to obtain a supernatant.

[0048] Among them, the final concentration of the formic acid is preferably 0.1 - 4% (such as 2%).

[0049] In Step 3, the conditions and operations of the extraction can be the conventional conditions and operations in the art, preferably oscillate at room temperature for 3 min, and then centrifuge at a speed of 2000 rpm for 5 min.

[0050] In step 3, the extraction solution for extraction can be acetonitrile and formic acid, and further, the final concentration is 50% acetonitrile + 0.1% formic acid.

[0051] In Step 3, the conditions and operations for concentration can be the conventional conditions and operations in the art. Vacuum concentration is preferred, and further centrifugal drying and concentration in a vacuum drying instrument are used. The temperature for concentration can be 30 - 80°C (for example, 60°C).

[0052] In Step 4, the conditions and operations for desalting can be the conventional conditions and operations in the art. The preferred conditions and operations are as shown in Table 1:

[0053]

[0054] In Step 4, after desalting the mixture obtained in Step 3, a concentration step may further be included. The conditions and operations for concentration can be the conventional conditions and operations in the art.

[0055] The present invention also provides a method for obtaining protein and / or peptide information, which includes the following steps:

[0056] Step 1: Obtain protein and / or peptide according to the above-mentioned method for obtaining protein and / or peptide.

[0057] Step 2: Perform mass spectrometry detection on the protein and / or peptide obtained in Step 1 to obtain the information of the protein and / or peptide. The mass spectrometry detection conditions are as follows:

[0058] Table 2

[0059]

[0060]

[0061] In the present invention, the term "success rate" refers to the frequency of obtaining high-quality detection results of tissue samples. High-quality detection of tissue samples means that the number of proteins obtained by single-needle mass spectrometry detection in 75 minutes is more than 4000, and the number of proteins obtained by single-needle mass spectrometry detection in 150 minutes is more than 5500, which is recorded as successful.

[0062] In the present invention, the term "protein groups" refers to the types / numbers of proteins obtained by matching the original data of the sample through mass spectrometry detection with the Maxquant protein library.

[0063] In the present invention, the term "peptide groups" refers to the number of peptides obtained by matching the original database of the sample through mass spectrometry detection with the Maxquant protein library.

[0064] In the present invention, the term "peptide matched spectrum" refers to the number of spectra matched by peptides after mass spectrometry detection of the sample, that is, the number of effective spectra.

[0065] In the present invention, the term "number of spectra" refers to all the spectra obtained by mass spectrometry scanning after a sample is detected by mass spectrometry.

[0066] In the present invention, the mass fraction is the mass percentage of the mass of the detergent in the total mass of each raw material.

[0067] In the present invention, the final concentration refers to the molar volume ratio of each component to the lysis solution.

[0068] In the present invention, the term "room temperature" refers to 10 - 30 °C.

[0069] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0070] The reagents and raw materials used in the present invention are all commercially available.

[0071] The positive and progressive effects of the present invention are as follows: The tissue samples treated with the lysis solution of the present invention have high yields of proteins and peptides, shorten the steps, reduce the possibility of contamination during the operation process, and improve the efficiency; moreover, the method of the present invention is particularly suitable for obtaining proteins and peptides from biopsy tissues or trace special tissue samples. Detailed implementation manners

[0072] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0073] Test instruments: Tandem liquid phase: EASY-nLC1200; Mass spectrometry instrument and model: Thermo QE-HFX.

[0074] Detection environment: Room temperature 21 °C, -22 °C, humidity 40% - 60%.

[0075] Identification of proteins:

[0076] (1) Obtain the original data of the sample after the mass spectrometry detection is completed;

[0077] (2) Upload the original data to maxquant for database searching; For maxquant database searching, all modification types are considered, and a fault tolerance matching function is added; The confidence level of the identified proteins is maintained above 99% (i.e., the mismatch rate is less than 1%); For the screening of the identified peptides, the confidence level is higher than 95%.

[0078] (3) Obtain the identification results, namely the number of proteins, the number of peptides, the number of spectra, etc.

[0079] Example 1

[0080] According to the formula of the lysis buffer in Table 3, the solid components were added to the liquid components for mixing, heated in a metal bath at 99 °C for 30 min, and then cooled to room temperature to obtain lysis buffers 1-8.

[0081] Table 3

[0082]

[0083]

[0084] Example 2

[0085] Tissue samples: 10 unstained white paraffin sections of normal human esophageal biopsy tissue with a length of 1 cm, a width of 10 μm, and a thickness of 10 μm were taken, and the tissue was scraped off, with a volume of about 0.5 μL, and placed in different test tubes.

[0086] Obtaining protein and peptide information:

[0087] Step 1: Lysing tissue samples

[0088] 50 μL of lysis buffers 1-8 (Lysis buffer) were added to each test tube respectively.

[0089] Step 2: Enzymatic digestion:

[0090] 3 μg of trypsin was added to each test tube, and the reaction was carried out with shaking in an incubator at 37 °C (1000 rpm) for 18 h.

[0091] Step 3: Removing DOC

[0092] 100% formic acid (FA) was added to each test tube to make the final concentration 2%, shaken at room temperature for 3 min, centrifuged at 12000 rpm for 5 min, and the supernatant was taken and transferred to a new EP tube.

[0093] Step 4: Extraction

[0094] 350 μL of extraction solution was added to each test tube, shaken at room temperature for 3 min, centrifuged at 1000 rpm for 5 min, and the supernatant was taken and transferred to a new EP tube.

[0095] Among them, the extraction solution formula: 50% acetonitrile (ACN) + 0.1% formic acid (FA), and the concentrations are all final concentrations.

[0096] Step 5: Vacuum drying

[0097] Centrifugal drying was carried out in a vacuum dryer under vacuum conditions at 60 °C.

[0098] Step 6: Desalting treatment (elution by centrifugation)

[0099] Column: T400 tip + 2 layers of C18 membrane

[0100] Procedure:

[0101] ① 100% ACN for 2 times, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0102] ② 50% ACN for 1 time, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0103] ③ 80% ACN for 1 time, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0104] ④ 50% ACN for 1 time, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0105] ⑤ 0.1% FA for 2 times, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0106] ⑥ loading for 2 times, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0107] ⑦ 0.1% FA for 2 times, centrifuge at 1000 g for 1 min, discard the collected liquid.

[0108] ⑧ Elution (50% ACN + 0.1% FA) for 2 times, centrifuge at 1000 g for 1 min, collect the eluted sample. 100 μL each time.

[0109] Step 7: Drying

[0110] Use a vacuum dryer to dry at 30 - 80 °C to obtain the protein and peptide segments.

[0111] Step 8: Mass spectrometry detection

[0112] Send the obtained protein and peptide segments to the mass spectrometry room for detection. Perform mass spectrometry detection according to the one - needle method (150 min). The detection conditions are shown in Table 4 below, and the detection results are shown in Table 5.

[0113] Table 4

[0114]

[0115]

[0116] Table 5

[0117]

[0118] Example 3

[0119] Take 10 white (unstained) paraffin sections of human colorectal biopsy tissue (in situ carcinoma) with a length of 1.5 cm, a width of 20 μm, and a thickness of 10 μm, scrape the tissue, and the volume is about 1 μL.

[0120] Obtain proteins and peptides and conduct detection according to the operation in Example 2 (wherein the mass spectrometry detection selects a 150-min gradient and the C18 analytical column is 30 cm long), and the detection results are shown in Table 6.

[0121] Table 6

[0122]

[0123] Example 4

[0124] Take 10 white (unstained) paraffin sections of human duodenal biopsy tissue (microinvasive carcinoma tissue) with a length of 1.5 cm, a width of 20 μm, and a thickness of 10 μm, scrape the tissue, and the volume is about 1 μL, and place them in different test tubes.

[0125] Obtain proteins and peptides and conduct detection according to the operation in Example 3, and the detection results are shown in Table 7.

[0126] Table 7

[0127]

[0128] Example 5

[0129] Take 10 white (unstained) paraffin sections of human gastric biopsy tissue (poorly differentiated tissue) with a length of 1.5 cm, a width of 20 μm, and a thickness of 10 μm, scrape the tissue, and the volume is about 1 μL, and place them in different test tubes.

[0130] Obtain proteins and peptides and conduct detection according to the operation in Example 2, and the detection results are shown in Table 8.

[0131] Table 8

[0132]

Claims

1. A lysis solution, which comprises: a detergent with a final concentration of 1-4% by mass; the detergent is sodium deoxycholate and / or ammonium bicarbonate; 3-(2-carboxy)phosphine hydrochloride with a final concentration of 5-20 μM; 5-chloro-anthranilic acid with a final concentration of 20-60 mM; tris(hydroxymethyl)aminomethane-hydrochloride with a final concentration of 50-200 mM; phenylmethylsulfonyl fluoride with a final concentration of 0.1-2 mM; and water; the lysis solution is a lysis solution for lysing tissue samples; the tissue samples are biopsy tissue samples or paraffin-embedded tissue samples.

2. The lysis solution according to claim 1, characterized in that, In the lysis solution, the detergent is sodium deoxycholate; and / or, in the lysis solution, the mass fraction of the detergent is 1-3%; and / or, in the lysis solution, the final concentration of the 3-(2-carboxy)phosphine hydrochloride is 10-20 μM; and / or, in the lysis solution, the final concentration of the 5-chloro-anthranilic acid is 20-40 mM; and / or, in the lysis solution, the final concentration of the tris(hydroxymethyl)aminomethane-hydrochloride is 50-100 mM; and / or, in the lysis solution, the final concentration of the phenylmethylsulfonyl fluoride is 1-2 mM.

3. The lysate according to claim 2, wherein In the lysis solution, the paraffin-embedded tissue samples are paraffin-embedded tissue samples of normal human esophageal biopsy tissues, paraffin-embedded tissue samples of human colorectal biopsy tissues, paraffin-embedded tissue samples of human duodenal biopsy tissues or paraffin-embedded tissue samples of human gastric biopsy tissues.

4. The lysis solution according to claim 3, characterized in that, The paraffin-embedded tissue samples of human colorectal biopsy tissues are paraffin-embedded tissue samples of in situ carcinoma; the paraffin-embedded tissue samples of human duodenal biopsy tissues are paraffin-embedded tissue samples of microinvasive carcinoma tissues; the paraffin-embedded tissue samples of human gastric biopsy tissues are preferably paraffin-embedded tissue samples of poorly differentiated tissues.

5. The lysis solution according to claim 1, wherein It comprises a detergent with a mass fraction of 1-3%, 3-(2-carboxy)phosphine hydrochloride with a final concentration of 10 μM, 5-chloro-anthranilic acid with a final concentration of 20 mM, tris(hydroxymethyl)methane-hydrochloride with a final concentration of 50 mM, phenylmethylsulfonyl fluoride with a final concentration of 2 mM and water; Or, it comprises a detergent with a mass fraction of 1-3%, 3-(2-carboxy)phosphine hydrochloride with a final concentration of 20 μM, 5-chloro-anthranilic acid with a final concentration of 40 mM, tris(hydroxymethyl)methane-hydrochloride with a final concentration of 100 mM, phenylmethylsulfonyl fluoride with a final concentration of 2 mM and water.

6. The lysate according to any one of claims 1-5, characterized in that, The lysis solution is composed of the detergent, the 3-(2-carboxy)phosphine hydrochloride, the 5-chloro-anthranilic acid, the tris(hydroxymethyl)aminomethane-hydrochloride, the phenylmethylsulfonyl fluoride and the water.

7. A method for preparing the lysis solution according to any one of claims 1-6, characterized in that, It comprises the following steps: Mix the components of the lysis solution, heat and cool to room temperature to obtain the lysis solution.

8. The preparation method according to claim 7, characterized in that, The temperature of the heating is 90-100 °C; The time of the heating is 5-60 min.

9. A kit, characterized in that, It comprises the lysis solution according to any one of claims 1-6 and at least one container for containing the lysis solution according to any one of claims 1-6.

10. Use of the lysis solution according to any one of claims 1-6 in obtaining proteins and / or peptides from tissue samples.

11. A method for obtaining proteins and / or peptides, characterized in that, It includes the following steps: Step 1: Lyse and denature the tissue sample in the lysis solution according to any one of claims 1-6 to obtain mixture 1; Step 2: Under the action of trypsin, enzymatically digest the mixture obtained in Step 1 to obtain mixture 2; Step 3: Remove the detergent from the mixture 2 obtained in Step 2, extract and concentrate it to obtain mixture 3; Step 4: Desalt the mixture obtained in Step 3 to obtain proteins and / or peptides.

12. The method according to claim 11, characterized in that, In Step 1, the tissue sample is the tissue sample as described in claim 2; And / or, in Step 1, the temperature of the lysis and denaturation is 90-100 °C; And / or, in Step 1, the time of the lysis and denaturation is 5-60 min; And / or, in Step 2, the dosage of trypsin is 40-60 μg / mL; And / or, in Step 2, the temperature of the enzymatic digestion is 30-40 °C; And / or, in Step 2, the time of the enzymatic digestion is 12-24 h; And / or, in Step 2, the oscillation speed of the enzymatic digestion is 300-1500 rpm; And / or, in Step 3, the method of removing the detergent is not adding a remover; And / or, in Step 3, the method of removing the detergent includes the following steps: under the presence of formic acid, oscillate for 1-5 min, centrifuge at a speed of 8000-12500 rpm to obtain the supernatant; And / or, in Step 3, the extraction conditions are to oscillate at room temperature for 3 min, and then centrifuge at a speed of 2000 rpm for 5 min; And / or, in Step 3, the extraction solution for extraction is acetonitrile and formic acid, and further is acetonitrile with a final concentration of 50% + 0.1% formic acid; And / or, in Step 3, the concentration is by vacuum concentration, and further is centrifugal drying and concentration in a vacuum drying instrument; And / or, in Step 4, the conditions and operations for desalting are as follows:

13. The method according to claim 12, wherein In Step 3, the remover is carbon powder; And / or, in Step 3, the final concentration of formic acid is 0.1-4%; And / or, in Step 3, the temperature of the concentration is 30-80 °C.

14. A method for obtaining protein and / or peptide information, characterized in that, It includes the following steps: Step 1: Obtain proteins and / or peptides according to the method for obtaining proteins and / or peptides according to any one of claims 11-13; Step 2: Perform mass spectrometry detection on the proteins and / or peptides obtained in Step 1 to obtain the information of the proteins and / or peptides, where the mass spectrometry detection conditions are as follows:

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