CN111751548A - Protein labeling method - Google Patents
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 101
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 101
- 238000002372 labelling Methods 0.000 title claims description 63
- 239000001963 growth medium Substances 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000004475 Arginine Substances 0.000 claims abstract description 8
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 8
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims abstract description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 8
- 102000008072 Lymphokines Human genes 0.000 claims abstract description 8
- 108010074338 Lymphokines Proteins 0.000 claims abstract description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000004472 Lysine Substances 0.000 claims abstract description 8
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 150000001413 amino acids Chemical class 0.000 claims abstract description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000006143 cell culture medium Substances 0.000 claims abstract description 8
- 239000003102 growth factor Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 42
- 238000001948 isotopic labelling Methods 0.000 claims description 14
- 238000005804 alkylation reaction Methods 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 10
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 9
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 9
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 9
- 239000001099 ammonium carbonate Substances 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 8
- WSFSSNUMVMOOMR-DICFDUPASA-N dideuteriomethanone Chemical compound [2H]C([2H])=O WSFSSNUMVMOOMR-DICFDUPASA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 238000004925 denaturation Methods 0.000 claims description 7
- 230000036425 denaturation Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000000751 protein extraction Methods 0.000 claims description 7
- 230000009467 reduction Effects 0.000 claims description 7
- 238000011946 reduction process Methods 0.000 claims description 7
- 238000011160 research Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 241001529936 Murinae Species 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 108090000317 Chymotrypsin Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 claims description 5
- 108010067372 Pancreatic elastase Proteins 0.000 claims description 5
- 102000016387 Pancreatic elastase Human genes 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 229960002376 chymotrypsin Drugs 0.000 claims description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 108010059339 submandibular proteinase A Proteins 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- KKTCWAXMXADOBB-UHFFFAOYSA-N azanium;hydrogen carbonate;hydrate Chemical compound [NH4+].O.OC([O-])=O KKTCWAXMXADOBB-UHFFFAOYSA-N 0.000 claims 1
- 238000007086 side reaction Methods 0.000 abstract description 9
- 239000000126 substance Substances 0.000 abstract description 6
- 230000002255 enzymatic effect Effects 0.000 abstract description 5
- 238000007796 conventional method Methods 0.000 abstract description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses a protein marking method, which comprises the following steps: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture; adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time; the method can complete the marking of the sample within dozens of minutes, has lower quality of the protein sample, further reduces the used cost, has the advantages of fewer reaction steps, no occurrence of serious side reaction, and realization of high-efficiency and accurate treatment under the condition of consuming less samples, and solves the problems that certain side reaction can be generated in the conventional methods for enzymatic marking and chemical marking of the protein, and the reaction steps are more, so that a large amount of samples are lost, and the marking efficiency and the identification accuracy are reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a protein labeling method.
Background
The main purpose of protein labeling is to monitor biological processes, to aid in detection (e.g., reliable quantification of compounds, specific detection of protein modifications), or to purify labeled proteins and their binding partners. The labeling of proteins can improve detection sensitivity and simplify the detection workflow. Large-scale identification and quantitative analysis of proteins is the key to proteomics research. The novel protein quantitative analysis technology plays a key promoting role in the discovery of basic life mechanisms and disease markers. Among them, stable isotope chemical labeling technology based on biological mass spectrometry plays an increasingly important role in the relative and absolute quantification of proteins.
The protein labeling method has the advantages that certain side reactions can be generated in the conventional methods for protein enzymatic labeling and chemical labeling, and a large number of reaction steps cause great loss of samples, so that the labeling efficiency and the identification accuracy are reduced.
SUMMARY OF THE PATENT FOR INVENTION
The invention aims to provide a protein marking method, which has the advantages of fewer reaction steps, no occurrence of serious side reaction and capability of realizing efficient and accurate treatment under the condition of consuming less samples, and solves the problems that certain side reaction is generated in the conventional enzymatic marking and chemical marking methods for protein, and the reaction steps are more, so that a large amount of samples are lost, and the marking efficiency and the identification accuracy are reduced.
In order to achieve the purpose, the invention provides the following technical scheme: a protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the medium to divide and proliferate the cells, collecting a part of the cells, and extracting proteins from the collected cells;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis treatment on the protein;
(5) labeling of the product: carrying out isotope labeling on the product after the enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent;
(6) and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
Preferably, in the step (3), the collected cells are cells which have undergone division and proliferation for more than 6 times, and the culture medium is equally divided into three parts during culture, so as to improve the accuracy of the test data.
Preferably, in the step (3), the extracted protein is derived from any one of a monoclonal antibody, a bispecific antibody, a murine antibody, a chimeric antibody, a human antibody or a humanized antibody.
Preferably, in the step (4), the whole process is carried out in ammonium bicarbonate aqueous solution, the mass ratio of the dissolved protein mixture is different, and the reagent added in the alkylation process is any one of dithiothreitol or iodoacetamide.
Preferably, in the step (4), the protease is any one or a combination of more than two of trypsin, specific endopeptidase Arg-C, Lys-C, chymotrypsin and elastase.
Preferably, in the step (4), the pH value of the ammonium bicarbonate solution used for enzymolysis is 8-9, the mass ratio of the protein to the enzyme is 125:1, the reaction time is 14-17h, and the reaction temperature is 37 ℃.
Preference is given toIn the step (5), the light labeling reagent may be CH2O、NaCNBH3And CH2O、NaCNBD3In any combination, the corresponding re-labeling agent may be CD2O、NaCNBD3And CD2O、NaCNBH3Any one of the combinations.
Preferably, in the step (5), the addition amount of the light labeling reagent is 0.005 to 1mol, the addition amount of the heavy labeling reagent is 0.005 to 1mol, and the two added solutions are reacted under the same reagent concentration for 50 to 70 min.
Preferably, in the step (6), the freeze-drying operation is performed by using a freeze-dryer model of Heto lyolab 3000 at a freezing temperature of-58 ℃.
Preferably, in the step (6), the buffer used in the LC-MS assay is any one or a combination of two or more of acetone, paraformaldehyde and PBS buffer.
Compared with the prior art, the invention has the following beneficial effects:
the method can complete the marking of the sample within dozens of minutes, has lower quality of the protein sample, further reduces the used cost, has the advantages of fewer reaction steps, no occurrence of serious side reaction, and realization of high-efficiency and accurate treatment under the condition of consuming less samples, and solves the problems that certain side reaction can be generated in the conventional methods for enzymatic marking and chemical marking of the protein, and the reaction steps are more, so that a large amount of samples are lost, and the marking efficiency and the identification accuracy are reduced.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments in the present invention patent, and it is obvious that the described embodiments are only a part of the embodiments of the present invention patent, and not all of the embodiments. All other embodiments obtained by a person skilled in the art based on the embodiments in the patent of the invention without any inventive work belong to the protection scope of the patent of the invention.
A protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the medium to divide and proliferate the cells, collecting a part of the cells, and extracting proteins from the collected cells;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis treatment on the protein;
(5) labeling of the product: carrying out isotope labeling on the product after the enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent;
(6) and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
The first embodiment is as follows:
a protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the culture medium, dividing and proliferating the cells, collecting part of the cells, and extracting protein from the collected cells, wherein the collected cells are the cells which are divided and proliferated for more than 6 times, and the culture medium is equally divided into three parts during culture so as to improve the accuracy of test data, and the extracted protein is derived from any one of monoclonal antibody, bispecific antibody, murine antibody, chimeric antibody, human antibody or humanized antibody;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis treatment on the protein;
(5) labeling of the product: carrying out isotope labeling on the product after the enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent;
(6) and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
Example two:
a protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the culture medium, dividing and proliferating the cells, collecting part of the cells, and extracting protein from the collected cells, wherein the collected cells are the cells which are divided and proliferated for more than 6 times, and the culture medium is equally divided into three parts during culture so as to improve the accuracy of test data, and the extracted protein is derived from any one of monoclonal antibody, bispecific antibody, murine antibody, chimeric antibody, human antibody or humanized antibody;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis on the protein, wherein the whole process is carried out in ammonium bicarbonate aqueous solution, the mass ratio of the dissolved protein mixture is different, a reagent added in the alkylation process is any one of dithiothreitol or iodoacetamide, the protease is any one or the combination of more than two of trypsin, specific endopeptidase Arg-C, Lys-C, chymotrypsin or elastase, the pH value of the ammonium bicarbonate solution used for enzymolysis is 8-9, the mass ratio of the protein to the enzyme is 125:1, the reaction time is 14-17h, and the reaction temperature is 37 ℃;
(5) labeling of the product: carrying out isotope labeling on the product after the enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent;
(6) and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
Example three:
a protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the culture medium, dividing and proliferating the cells, collecting part of the cells, and extracting protein from the collected cells, wherein the collected cells are the cells which are divided and proliferated for more than 6 times, and the culture medium is equally divided into three parts during culture so as to improve the accuracy of test data, and the extracted protein is derived from any one of monoclonal antibody, bispecific antibody, murine antibody, chimeric antibody, human antibody or humanized antibody;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis on the protein, wherein the whole process is carried out in ammonium bicarbonate aqueous solution, the mass ratio of the dissolved protein mixture is different, a reagent added in the alkylation process is any one of dithiothreitol or iodoacetamide, the protease is any one or the combination of more than two of trypsin, specific endopeptidase Arg-C, Lys-C, chymotrypsin or elastase, the pH value of the ammonium bicarbonate solution used for enzymolysis is 8-9, the mass ratio of the protein to the enzyme is 125:1, the reaction time is 14-17h, and the reaction temperature is 37 ℃;
(5) labeling of the product: carrying out isotope labeling on the product after enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent, wherein the light labeling reagent can be CH2O、NaCNBH3And CH2O、NaCNBD3In any combination, the corresponding re-labeling agent may be CD2O、NaCNBD3And CD2O、NaCNBH3In any one of the combinations, the addition amount of the light labeling reagent is 0.005-1mol, the addition amount of the heavy labeling reagent is 0.005-1mol, and the two added solutions react under the same reagent concentration for 50-70 min;
(6) and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
Example four:
a protein labeling method, which comprises the following steps:
(1) and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
(2) preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
(3) protein extraction: adding the cells to the culture medium, dividing and proliferating the cells, collecting part of the cells, and extracting protein from the collected cells, wherein the collected cells are the cells which are divided and proliferated for more than 6 times, and the culture medium is equally divided into three parts during culture so as to improve the accuracy of test data, and the extracted protein is derived from any one of monoclonal antibody, bispecific antibody, murine antibody, chimeric antibody, human antibody or humanized antibody;
(4) protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis on the protein, wherein the whole process is carried out in ammonium bicarbonate aqueous solution, the mass ratio of the dissolved protein mixture is different, a reagent added in the alkylation process is any one of dithiothreitol or iodoacetamide, the protease is any one or the combination of more than two of trypsin, specific endopeptidase Arg-C, Lys-C, chymotrypsin or elastase, the pH value of the ammonium bicarbonate solution used for enzymolysis is 8-9, the mass ratio of the protein to the enzyme is 125:1, the reaction time is 14-17h, and the reaction temperature is 37 ℃;
(5) labeling of the product: carrying out isotope labeling on the product after enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent, wherein the light labeling reagent can be CH2O、NaCNBH3And CH2O、NaCNBD3In any combination, the corresponding re-labeling agent may be CD2O、NaCNBD3And CD2O、NaCNBH3In any one of the combinations, the addition amount of the light labeling reagent is 0.005-1mol, the addition amount of the heavy labeling reagent is 0.005-1mol, and the two added solutions react under the same reagent concentration for 50-70 min;
(6) and (3) treating a product: and (2) freeze-drying the products marked by two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative study on the protein, wherein the freeze-drying operation of the products adopts a freeze-drying instrument, the model of the freeze-drying instrument is Heto lyolab 3000, the freezing temperature is-58 ℃, and the buffer solution used for the liquid chromatography-mass spectrometry analysis is any one or the combination of more than two of acetone, paraformaldehyde and PBS buffer solution.
The method can complete the marking of the sample within dozens of minutes, has lower quality of the protein sample, further reduces the used cost, has the advantages of fewer reaction steps, no occurrence of serious side reaction, and realization of high-efficiency and accurate treatment under the condition of consuming less samples, and solves the problems that certain side reaction can be generated in the conventional methods for enzymatic marking and chemical marking of the protein, and the reaction steps are more, so that a large amount of samples are lost, and the marking efficiency and the identification accuracy are reduced.
Although embodiments of the present patent have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the present patent, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A protein labeling method characterized by: the marking method comprises the following steps:
and (3) culturing the cells: firstly, extracting cell factors from lymphokines or growth factors, and then selecting a culture medium to place cells in the culture medium for culture;
preparation of a culture medium: adding arginine, lysine and proline to a cell culture medium lacking amino acids, uniformly mixing them and standing for a period of time;
protein extraction: adding the cells to the medium to divide and proliferate the cells, collecting a part of the cells, and extracting proteins from the collected cells;
protein treatment: respectively carrying out protein denaturation, protein reduction and alkylation processes on the protein extract, and then carrying out enzymolysis treatment on the protein;
labeling of the product: carrying out isotope labeling on the product after the enzymolysis treatment, and respectively carrying out isotope labeling by adopting a light labeling reagent and a heavy labeling reagent;
and (3) treating a product: and (3) freeze-drying the products marked by the two different reagents after reaction, and performing liquid chromatography-mass spectrometry analysis to perform quantitative research on the protein.
2. The method for protein labeling according to claim 1, wherein: in the step (3), the collected cells are cells which are divided and proliferated for more than 6 times, and the culture medium is equally divided into three parts during culture so as to improve the accuracy of test data.
3. The method for protein labeling according to claim 1, wherein: in the step (3), the extracted protein is derived from any one of a monoclonal antibody, a bispecific antibody, a murine antibody, a chimeric antibody, a human antibody or a humanized antibody.
4. The method for protein labeling according to claim 1, wherein: in the step (4), the whole process is carried out in ammonium bicarbonate water solution, the mass ratio of the dissolved protein mixture is different, and the reagent added in the alkylation process is any one of dithiothreitol or iodoacetamide.
5. The method for protein labeling according to claim 1, wherein: in the step (4), the protease is any one or a combination of more than two of trypsin, specific endopeptidase Arg-C, Lys-C, chymotrypsin or elastase.
6. The method for protein labeling according to claim 4, wherein: in the step (4), the pH value of the ammonium bicarbonate solution used for enzymolysis is 8-9, the mass ratio of protein to enzyme is 125:1, the reaction time is 14-17h, and the reaction temperature is 37 ℃.
7. The method for protein labeling according to claim 1, wherein: in the step (5), the light labeling reagent may beIs CH2O、NaCNBH3And CH2O、NaCNBD3In any combination, the corresponding re-labeling agent may be CD2O、NaCNBD3And CD2O、NaCNBH3Any one of the combinations.
8. The method for protein labeling according to claim 1, wherein: in the step (5), the addition amount of the light labeling reagent is 0.005-1mol, the addition amount of the heavy labeling reagent is 0.005-1mol, and the two added solutions react under the same reagent concentration for 50-70 min.
9. The method for protein labeling according to claim 1, wherein: in the step (6), the freeze-drying operation of the product is carried out by using a freeze-dryer which is a type of Heto lyolab 3000, and the freezing temperature is-58 ℃.
10. The method for protein labeling according to claim 1, wherein: in the step (6), the buffer solution used in the LC-MS analysis is any one or a combination of two or more of acetone, paraformaldehyde and PBS buffer solution.
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