CN113527406B - Method for extracting protein from formalin-fixed paraffin-embedded sample - Google Patents
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Abstract
The invention belongs to the technical field of biochemical analysis, and particularly relates to a method for extracting protein from a formalin-fixed paraffin-embedded sample, which comprises the following steps: heating formalin-fixed paraffin-embedded samples at a slightly alkaline pH value at a temperature of 90-100 ℃ for a period of time; adding protease into the obtained heating treatment liquid, and carrying out enzymolysis treatment for a period of time under the proper action temperature range of the protease; and centrifuging the obtained enzymolysis liquid to obtain supernatant containing the enzymolysis peptide section of the protein. The method of the invention realizes the decrosslinking and denaturation of the protein through the heating treatment simultaneously, can denature the protein without adding a chemical denaturant, can omit the operation of correcting the pH value to remove the chemical denaturant, can omit the subsequent precipitation and desalting operation, is beneficial to simplifying experimental operation, especially batch sample operation and reduces the loss of required peptide fragment samples.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis, and particularly relates to a method for extracting proteins from a formalin-fixed paraffin-embedded sample.
Background
Formalin-fixed paraffin embedding (FFPE) technology is a common way to preserve biopsy and surgical specimens. FFPE specimens can be stored at room temperature, avoiding the complexity and risk of cryopreservation. When an FFPE specimen is subsequently required for analysis, an FFPE sample, e.g., a sample comprising nucleic acids and/or proteins, can be obtained from the FFPE specimen and subjected to the required analysis by a nucleic acid analysis method or a protein analysis method. However, although formaldehyde, the major component of formalin fixatives, can make samples more stable for storage, it also causes extensive cross-linking and adducts of nucleic acids, proteins and other biomolecules within the sample, affecting subsequent analysis. Therefore, prior to analyzing a sample obtained from an FFPE specimen, the sample needs to be processed in order to perform the analysis operation of interest. For example, in the case of analyzing proteins in FFPE samples by a protein mass spectrometry method, it is necessary to first crosslink and denature the crosslinked proteins and then cleave the peptides to perform protein mass spectrometry.
Typically, obtaining a small sample of FFPE from an FFPE specimen can meet the needs of the analysis. A small sample of FFPE may be cut from the FFPE specimen using a blade or the like. A more advanced approach is to employ laser capture microdissection (laser capture microdissection, LCM) technology. The technology can selectively capture, separate and purify a single type of target cell group or single target cell from a tissue slice or a cell smear under a microscope on the premise of not damaging a tissue structure and preserving the integrity of cells to be captured and surrounding tissue morphology. LCM technology successfully addresses the problem of cellular heterogeneity in tissues and is a revolutionary technique for molecular pathology and oncology research. Proteomic analysis has been widely used in various aspects of disease research, but tissue heterogeneity plagues analysis of research results. LCM technology has become an important tool for proteomic research because it allows for the direct acquisition of target cells from tissue sections (e.g., paraffin-embedded sections and frozen sections). The FFPE specimen is cut by LCM technology, so that an FFPE-LCM sample containing cells or cell groups of interest can be obtained, and further, nucleic acid or protein extraction is carried out on the FFPE-LCM sample, and the FFPE-LCM sample can be used for genomics or proteomics research. For protein extraction of FFPE-LCM samples, the amount of protein is increased, e.g., by using multiple FFPE-LCM samples, to extract enough protein for analysis [1] Or using thicker tissue sections [2] To perform protein extraction. With the finer demands on samples, FFPE-LCM samples are smaller and smaller in area, and contain only about 2500 cells at a minimum, thus placing higher demands on the extraction of trace proteins.
Literature [1-5] Comparative experiments performed by screening several commonly used protein denaturation extraction reagents and then performing a minification-system protein extraction on FFPE-LCM samples are reported. Rapid SF is a denaturant with the best extraction effect on trace protein samples, which is found by the current comparison experiment [3] . Experimental technology for extracting protein by adopting RapiGest SFThe process involves heat treatment of the sample to crosslink the protein, then adding rapidest SF to denature the protein, then adjusting to below pH 2 to remove rapidest SF, then adjusting to pH7.4 to reduce/alkylate/reduce, then adding trypsin to treat, and then precipitating centrifugation and desalting the trace peptide to obtain a sample of protein peptide for analysis such as protein mass spectrometry. Because of the use of rapidest SF, the experimental technological process needs to perform pH correction on the trace volume sample, which is not beneficial to batch sample operation, and further needs to perform precipitation centrifugation and desalting on trace peptide fragments, which can cause too high loss rate for trace samples.
Disclosure of Invention
The invention aims to provide a novel protein extraction method which is suitable for extracting proteins from Formalin Fixed Paraffin Embedded (FFPE) samples, is particularly suitable for extracting proteins from FFPE-LCM samples, and can solve the problems that pH correction in the prior art is not beneficial to batch sample operation and loss is caused by precipitation centrifugation and desalination of trace peptide fragments.
Specifically, the aim of the invention is achieved by the following technical scheme:
a method of extracting protein from an FFPE sample, the method comprising the steps of:
(1) Heating FFPE sample at 90-100deg.C for 10-60 min under weak alkaline pH value;
(2) Adding protease into the obtained heating treatment liquid, and carrying out enzymolysis treatment for 2-8 hours at a proper action temperature range of the protease;
(3) And centrifuging the obtained enzymolysis liquid to obtain supernatant containing the enzymolysis peptide section of the protein.
In the method, the FFPE sample is a sample obtained from an FFPE specimen. Preferably, the FFPE sample is a sample obtained from FFPE specimen by Laser Capture Microdissection (LCM) technique, abbreviated as FFPE-LCM sample.
Further, in step (1), the weakly alkaline pH is from pH 7.0 to 8.5. Preferably, the slightly alkaline pH is within a suitable pH range for the protease.
Further, the weakly alkaline pH is achieved by adding a weakly alkaline solution to the FFPE sample. Preferably, the weight-to-volume ratio of the weight of the FFPE sample to the volume of the weakly alkaline solution is 1 (5-50).
Further, the weakly basic solution is an ammonium bicarbonate solution or a tetraethylammonium bromide (TEAB) solution. Still further, the concentration of the weakly basic solution is 25-500mM.
Further, in step (1), the heating time was 20 minutes.
Further, in the step (2), the ratio of the weight of the heat treatment liquid to the weight of the protease is 1 (0.1-0.5).
Further, in the step (2), the time of the enzymolysis treatment is 2 hours.
Further, in step (2), the protease is trypsin or rLysC protease (which is a lysine protease). Preferably, the protease is trypsin.
Further, in the step (2), an organic solvent may be optionally added to promote the enzymolysis treatment. Preferably, the organic solvent is acetonitrile or methanol. In general, the organic solvent is added in an amount of 5 to 30% based on the heat treatment liquid.
Preferably, the method employs FFPE-LCM samples, comprising the steps of:
(1) To a trace of FFPE-LCM sample, 10. Mu.L of 50mM ammonium hydroxide solution pH 8.1 was added, followed by heat treatment at 95℃for 20 minutes;
(2) Adding 0.1 mug trypsin into the obtained heating treatment liquid, and carrying out enzymolysis treatment for 2 hours at 37 ℃;
(3) And centrifuging 12000-30000g of the obtained enzymolysis liquid for 5-10 minutes at normal temperature to obtain supernatant of the enzymolysis peptide section containing the protein.
Still more preferably, in the above step (2), acetonitrile is added at a final concentration of 5 to 30% to promote the enzymolysis.
The supernatant of the enzymatic peptide fragment comprising the protein obtained in step (3) may be used for protein mass spectrometry or may be detected by other means.
The invention has the beneficial effects that:
the method of the invention realizes the decrosslinking and denaturation of the protein through the heating treatment at the same time, can denature the protein without adding a chemical denaturant (such as a Rapid SF reagent), can omit the operation of correcting the pH to remove the chemical denaturant, can omit the subsequent precipitation and desalting operation, is beneficial to simplifying experimental operation, especially batch sample operation, and reduces the loss of required peptide fragment samples.
Drawings
FIG. 1 shows a 1-hour mass spectrometry plot of the supernatant of a first human kidney FFPE-LCM protein of example 1 of the present invention;
FIG. 2 shows a 1-hour mass spectrometry plot of the supernatant of the first human kidney FFPE-LCM protein of example 1 of the present invention;
FIG. 3 shows a 2-hour mass spectrometry plot of the supernatant of the first mouse liver FFPE protein in example 2 of the present invention;
figure 4 shows a 2 hour mass spectrometry plot of the supernatant of the first mouse liver FFPE protein in example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments. All scientific terms and terminology used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined.
As described above, the present invention provides a method for extracting proteins from formalin-fixed paraffin-embedded samples.
The term "formalin-fixed paraffin-embedded specimen" as used herein refers to FFPE specimens for short, and refers to biological specimens, including but not limited to cell specimens, tissue specimens, organ specimens, fixed with formalin and embedded with paraffin. The FFPE specimen can realize long-term preservation of biological samples at normal temperature.
The term "formalin-fixed paraffin-embedded sample" as used herein refers to FFPE samples for short, meaning samples obtained from preserved FFPE samples for performing detection assays, typically in small amounts sufficient for performing detection assays. Typically, FFPE specimens can be cut from FFPE specimens with a tool such as a blade. A more advanced approach is to cut FFPE samples from FFPE specimens using Laser Capture Microdissection (LCM) techniques, where the obtained samples are simply "FFPE-LCM samples".
The FFPE sample is subjected to formaldehyde crosslinking, so that the amino acid at the position where the protein in the sample can be cut by the protease is subjected to covalent modification, and the cleavage of the protease is affected. Therefore, it is necessary to crosslink the protein first and then carry out proteolysis. But also protein denaturants are often used to further open the protein structure for enzymatic treatment. However, conventional chemical denaturants interfere with protein mass spectrometry detection, thus adding additional processing steps to the experiment, resulting in cumbersome experimental operations.
The problems of the prior art are further described by taking the denaturant RapiGest SF with the best extraction effect on the trace protein sample at present as an example. Rapidest SF is a protein denaturing agent that can open the protein structure and expose cleavage sites for subsequent proteolytic cleavage. Rapidest SF is a chemically labile surfactant that is extremely easily hydrolyzed under acidic conditions, and this unique property can be exploited to remove it from solution when needed. Protein extraction experimental technological process using rapidest SF [3] The following is provided. First, the sample is sonicated for 10 minutes to remove bubbles in the sample. The samples were shaken at 99℃and 800rpm for 1 hour. Cooled to room temperature, 0.1% rapidest SF was added to a final concentration of 0.01%, shaking was continued at 800rpm for 10 minutes, and then sonication was performed for 10 minutes to remove bubbles. Then, the pH was adjusted to 7.4, and reduction/alkylation/reduction was performed to control the pH to 7.4. Then, trypsin was used at 60. Mu.g/ml in 50mM NH 4 HCO 3 For 16 hours followed by treatment with 30. Mu.g/ml trypsin in 80% Acetonitrile (ACN) for 3 hours. The reaction was quenched by the addition of 0.5% trifluoroacetic acid (TFA) and shaken at 600rpm at 37℃for 45 minutes. Then, the supernatant was collected by centrifugation at 16000RCF (relative centrifugal force) for 10 minutes and evaporated in vacuo. 2. Mu.g of protein was concentrated with ZipTip pipette tip and then with 9. Mu.L of 100mM NH 4 HCO 2 And (3) reconstructing, and adding MDPS Mix to prepare a protein peptide fragment sample for protein mass spectrometry analysis.
In the experimental technological process, after the protein is denatured by adopting the Rapid SF, the pH value is adjusted to 7.4 for reduction/alkylation/reduction, trypsin treatment is added, and then precipitation centrifugation and desalting are further needed for trace peptide fragments, so that a protein peptide fragment sample for protein mass spectrometry analysis is prepared. The pH correction is carried out on the micro-volume sample, which is not beneficial to batch sample operation; and the trace peptide fragments are subjected to precipitation centrifugation and desalination, so that the loss rate of the trace samples is too high.
In view of the above technical problems, the present inventors have conducted intensive studies and have recognized that the decrosslinking of proteins is essentially a way to break crosslinked covalent bonds by heating, which is a way of denaturing proteins. Thus, the present inventors innovatively propose that protein denaturation can be achieved without adding a chemical agent, but directly by heating to effect proteolytic crosslinking and denaturation, so that the subsequent step of removing the chemical agent can be omitted. Thus, the present inventors have developed the present invention.
In particular, the present invention provides a method of extracting protein from an FFPE sample, the method comprising the steps of:
(1) Heating FFPE sample at 90-100deg.C for 10-60 min under weak alkaline pH value;
(2) Adding protease into the obtained heating treatment liquid, and carrying out enzymolysis treatment for 2-8 hours at a proper action temperature range of the protease;
(3) And centrifuging the obtained enzymolysis liquid to obtain supernatant containing the enzymolysis peptide section of the protein.
In a specific embodiment of the invention, the FFPE sample is a sample obtained from an FFPE specimen. In a preferred embodiment of the present invention, the FFPE sample is a sample obtained by a Laser Capture Microdissection (LCM) technique, abbreviated as FFPE-LCM sample.
The term "weakly alkaline pH" as used herein refers to alkaline pH conditions in the range of pH 7.0-8.5, such as pH 7.1, pH 7.2, pH 7.3, pH7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5.
In a specific embodiment of the invention, in step (1), the weakly basic pH is achieved by adding a weakly basic solution to the FFPE sample.
In a preferred embodiment of the invention, the pH of the weakly basic solution is in the pH range suitable for the protease, so that the enzymatic treatment can be carried out without adjusting the pH in step (2) by directly adding the protease.
In a specific embodiment of the present invention, if the pH of the weakly basic solution added in step (1) is not within the range of the pH suitable for the protease, the resulting heat-treated liquid may be adjusted to the range of the pH suitable for the protease by adding an acid or a base in step (2).
In particular embodiments of the invention, the weight-to-volume ratio of the weight of the FFPE sample to the volume of the weakly alkaline solution is 1 (5-50), such as 1:5, 1:10, 1:20, 1:30, 1:40, or 1:50. Typically, the FFPE samples are between 5-10 μm thick. If the FFPE sample is 0.01-1cm in area 2 The volume of the weakly alkaline solution is taken to be 10-50. Mu.L. For easy operation, 0.1cm in area 2 The following sections were all prepared with 10. Mu.L of a weakly alkaline solution. If the FFPE sample is an FFPE-LCM sample, 10. Mu.L of a weakly alkaline solution is typically used.
Weakly basic solutions having a pH within the pH range suitable for the protease are well known in the art or readily determinable by those skilled in the art. In a specific embodiment of the invention, the weakly basic solution is an ammonium bicarbonate solution or tetraethylammonium bromide (TEAB). Still further, the concentration of the weakly basic solution is 25-500mM.
In a specific embodiment of the present invention, in step (1), the heat treatment may be performed with a water bath or a metal bath.
In a specific embodiment of the invention, in step (1), the heating time is from 10 to 60 minutes, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 minutes, preferably 20 minutes.
In a specific embodiment of the invention, in step (2), the ratio of the weight of the heat treatment liquid to the weight of the protease is 1 (0.1-0.5), such as 1:0.1, 1:0.2, 1:0.3, 1:0.4 and 1:0.5.Typically, if the FFPE sample is 0.01-1cm in area 2 Adding 0.1-0.5 μg trypsin, if the area is 0.1cm 2 In the following sections, 0.1. Mu.g of trypsin was added.
In a specific embodiment of the invention, in step (2), the time of the enzymatic treatment is 2-8 hours, for example 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 hours, preferably 2 hours.
In a specific embodiment of the invention, in step (2), the protease is trypsin or rLysC protease (which is a lysine protease). Preferably, the protease is trypsin. Suitable temperature ranges for the protease are well known in the art, e.g. trypsin is suitable for a range of 35-40℃and 37℃is optimal for a range of action. In addition, the pH value of trypsin is pH 8.1.
In a specific embodiment of the present invention, in step (2), an organic solvent may be further added to promote the enzymatic hydrolysis treatment. Preferably, the organic solvent is acetonitrile or methanol. In general, the organic solvent is added in an amount of 5 to 30% based on the heat treatment liquid.
In a preferred embodiment of the present invention, the method employs FFPE-LCM samples, comprising the steps of:
(1) To a trace of FFPE-LCM sample, 10. Mu.L of 50mM ammonium hydroxide solution pH 8.1 was added, followed by heat treatment at 95℃for 20 minutes;
(2) Adding 0.1 mug trypsin into the obtained heating treatment liquid, and carrying out enzymolysis treatment for 2 hours at 37 ℃;
(3) And centrifuging 12000-30000g of the obtained enzymolysis liquid for 5-10 minutes at normal temperature to obtain supernatant of the enzymolysis peptide section containing the protein.
Still more preferably, in the above step (2), acetonitrile is added at a final concentration of 5 to 30% to promote the enzymolysis.
It should be noted that the actual operation of step (1) of the method of the present invention is usually performed in a sealed container such as a capped cuvette (e.g., a PCR cuvette). During heating, liquid (e.g., water) may evaporate and condense on the lid and/or walls of the container. Thus, if the heating time exceeds 20 minutes, it is necessary to take out the container every 20 minutes for instantaneous centrifugation to centrifuge off the liquid evaporated and condensed on the lid and/or wall of the container, and then to continue the heating process for the remaining time. Thus, the influence on the subsequent proteolysis caused by dehydration, drying, heating and coking of the sample can be avoided.
By the method of the invention, the supernatant of the enzymatic peptide fragment comprising the protein obtained in step (3) can be used for protein mass spectrometry detection, or detected by other means.
The invention is further illustrated by the following non-limiting examples. It should be noted that these embodiments are exemplary and are not intended to limit the invention in any way.
Example 1:
the FFPE specimens were obtained from human kidney tissue by Laser Capture Microdissection (LCM) method by puncturing FFPE specimens with a cell number of about 500-2000, placed on the lid of a 0.2ml EP centrifuge tube, and not visible to the naked eye. The lid was closed to the EP centrifuge tube, the sample was transferred to the bottom of the EP centrifuge tube, and the EP centrifuge tube was placed in a palm centrifuge and centrifuged at 20000g for 10s.
mu.L of 50mM pH 8.1 ammonium bicarbonate solution was added with a 10. Mu.L pipette and the EP centrifuge tube was directly placed on a 95℃metal bath and heated for 20 minutes.
The EP centrifuge tube was removed, 1. Mu.L of trypsin solution at a concentration of 0.1. Mu.g/. Mu.L was added, and then a fresh-keeping film was wrapped on the tube mouth, and the mixture was subjected to shaking enzymolysis at 37℃for 2 hours at 800rpm in Eppendorf ThermoMixture.
The EP centrifuge tube was removed, centrifuged at 20000g for 10 minutes at room temperature, the supernatant was taken and the pellet was discarded. The obtained supernatant contains FFPE-LCM sample protein, and can be used for protein mass spectrometry analysis or other analysis.
Example 2:
2 samples of FFPE were obtained from a sample of mouse liver tissue FFPE using a Laser Capture Microdissection (LCM) method, with a slice area of about 0.5 square cm, and placed on a slide.
10. Mu.L of 50mM ammonium bicarbonate pH 8.1 solution was added with a 10. Mu.L pipette, dropped onto a dewaxed and hydrated FFPE slide, then the slide was carefully scraped off with a 10. Mu.L tip, suspended in the drop, pipetted together into a 0.2ml EP centrifuge tube, and the EP centrifuge tube was placed on a 95℃metal bath and heated for 20 minutes.
The EP centrifuge tube was removed, placed in a palm centrifuge for instantaneous centrifugation, the liquid evaporated and condensed on the tube cap was centrifuged to the bottom of the tube to cover the FFPE sample, and then the EP centrifuge tube was placed back on the 95 ℃ metal bath for continued heating for 20 minutes.
The EP centrifuge tube was removed, 1. Mu.L of trypsin solution at a concentration of 0.25. Mu.g/. Mu.L was added, and then a fresh-keeping film was wrapped on the tube mouth, and the mixture was subjected to shaking enzymolysis at 37℃for 2 hours at 800rpm on Eppendorf TheroMixture.
The EP centrifuge tube was removed, centrifuged at 20000g for 10 minutes at room temperature, the supernatant was taken and the pellet was discarded. The obtained supernatant contains FFPE-LCM sample protein, and can be used for protein mass spectrometry analysis or other analysis.
Test example:
the supernatant obtained in example 1, which contains FFPE-LCM sample proteins, was obtained from 2 traces of human kidney tissue FFPE-LCM samples (about 500 to 2000 cells). Mass spectrometry was performed on a protein mass spectrometer (QE-HFX, thermo) using the two supernatants to identify 1218 proteins (fig. 1) and 1238 proteins (fig. 2), respectively, for 1 hour. And have been reported in the literature [3] Less than 900 proteins were identified from human kidney FFPE-LCM samples of about 2500 cells using a detection technique with multiple reagent types and more complex procedures.
The supernatant obtained in example 2 containing FFPE sample proteins was derived from FFPE section samples (about 0.5 square cm in area) of 2 mice liver tissue. Mass spectrometry was performed on a protein mass spectrometer (QE-HFX, thermo) using the two supernatants to identify 1521 proteins (fig. 3) and 1569 proteins (fig. 4), respectively, for 2 hours.
Summarizing:
the preparation of FFPE-LCM samples and FFPE sample proteins can be completed in less than 2.5 hours by using the method provided by the invention, and the experiment is rapid, the operation is simple and almost no peptide fragment is lost. The test results show that more proteins can be identified from a smaller cell number using the methods of the invention. Therefore, the method is hopeful to obtain the most protein peptide fragment samples from the trace clinical FFPE-LCM sample and FFPE sample for subsequent precise experimental analysis, and has great application value for more precise clinical detection marker discovery and disease mechanism research.
Reference is made to:
1) Nirmalan, n.j., hughes, c., peng, j., mcKenna, t., langridge, j., cairns, d.a., … Banks, r.e. (2011) Initial development and validation of a novel extraction method for quantitative minutes ing of the formalin-fixedd, paramffin-embedded tissue proteome for biomarker indexes, journal of Proteome Research,10 (2), 896-905.
2)
M.C.,Fahrner,M.,Oria,V.O.,Kühs,M.,Biniossek,M.L.,Werner,M.,…Schilling,O.(2018).Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization.Clinical Proteomics,15(1).
3)Longuespée,R.,Alberts,D.,Pottier,C.,Smargiasso,N.,Mazzucchelli,G.,Baiwir,D.,De Pauw,E.(2016).A laser microdissection-based workflow for FFPE tissue microproteomics:Important considerations for small sample processing.Methods,104,154–162.
4)Azimi,A.,Kaufman,K.L.,Ali,M.,Kossard,S.,&Fernandez-Penas,P.(2016).In silico analysis validates proteomic findings of formalin-fixed paraffin embedded cutaneous squamous cell carcinoma tissue.Cancer Genomics and Proteomics,13(6),453–466.
5)
J.R.,Ostasiewicz,P.,&Mann,M.(2011).High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers.Journal of Proteome Research,10(7),3040–3049.
The invention has been described with particular reference to the examples which are intended to be illustrative of the invention and not limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art according to the idea of the invention. Such deductions, modifications or alternatives fall within the scope of the claims of the present invention.
Claims (15)
1. A method of extracting protein from a formalin fixed paraffin embedded sample, the method comprising the steps of:
(1) Heating the formalin-fixed paraffin-embedded sample at a weak alkaline pH value of 7.1-8.5 for 10-60 minutes at a temperature of 90-100 ℃;
(2) Adding protease into the obtained heat treatment liquid, and carrying out enzymolysis treatment for 2-8 hours at a proper action temperature range of the protease, wherein the protease is trypsin or rLysC protease;
(3) And centrifuging the obtained enzymolysis liquid to obtain supernatant containing the enzymolysis peptide section of the protein.
2. The method of claim 1, wherein the slightly alkaline pH is within a suitable pH range for the protease.
3. The method of claim 2, wherein in step (1), the weakly basic pH is achieved by adding a weakly basic solution to the formalin fixed paraffin embedded sample.
4. The method of claim 3, wherein the ratio of the weight of the formalin-fixed paraffin embedded sample to the volume of the weakly alkaline solution is 1 (5-50) by weight to volume.
5. A method according to claim 3, wherein in step (1) the weakly basic solution is an ammonium bicarbonate solution or a tetraethylammonium bromide solution.
6. The method of claim 5, wherein the weakly basic solution has a concentration of 25-500mM.
7. The method of claim 1, wherein in step (1), the heating time is 20 minutes.
8. The method according to claim 1, wherein in the step (2), the ratio of the weight of the heat treatment liquid to the weight of the protease is 1 (0.1-0.5).
9. The method according to claim 1, wherein in step (2), the time of the enzymatic hydrolysis treatment is 2 hours.
10. The method of claim 1, wherein in step (2), the protease is trypsin.
11. The method according to claim 1, wherein in step (2), an organic solvent is added to promote the enzymatic hydrolysis.
12. The method according to claim 11, wherein the organic solvent is acetonitrile or methanol.
13. The method according to claim 11, wherein the organic solvent is added in an amount of 5 to 30% based on the heat treatment liquid.
14. The method of claim 1, wherein the formalin-fixed paraffin-embedded sample is a sample obtained from a formalin-fixed paraffin-embedded specimen by a laser capture microdissection technique, the method comprising the steps of:
(1) Adding 10 mu L of 50mM ammonium hydroxide solution with pH of 8.1 into the trace sample, and then heating at 95 ℃ for 20 minutes;
(2) Adding 0.1 mug trypsin into the obtained heating treatment liquid, and performing enzymolysis treatment for 2 hours at 37 ℃;
(3) And centrifuging 12000-30000g of the obtained enzymolysis liquid for 5-10 minutes at normal temperature to obtain supernatant of the enzymolysis peptide section containing the protein.
15. The method according to claim 14, wherein acetonitrile is added in the final concentration of 5-30% in step (2) to promote the enzymolysis.
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| CN1803828A (en) * | 2005-12-28 | 2006-07-19 | 南方医科大学 | Method for extracting protein from paraffin-embedded tissue |
| CN102146112A (en) * | 2011-01-25 | 2011-08-10 | 天根生化科技（北京）有限公司 | Method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues |
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| CN1803828A (en) * | 2005-12-28 | 2006-07-19 | 南方医科大学 | Method for extracting protein from paraffin-embedded tissue |
| CN102146112A (en) * | 2011-01-25 | 2011-08-10 | 天根生化科技（北京）有限公司 | Method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues |
| CN110864939A (en) * | 2018-08-27 | 2020-03-06 | 深圳大学 | Protein extraction method and application |
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