Semi-quantitative evaluation method for substitution degree of non-protein amino acid
By synthesizing non-protein amino acid-engineered proteins in phenylalanine-deficient E.coli cells and using LC/MS-MS technology for semi-quantitative evaluation, the technical problem of assessing the degree of non-protein amino acid substitution in macromolecular proteins was solved, and effective assessment of macromolecular proteins was achieved.
Patent Information
- Application Number
- CN202411905053.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art cannot effectively assess the degree of substitution of non-protein amino acids in macromolecular proteins.
By expressing the recombinant plasmid in phenylalanine-deficient E.coli cells, non-protein amino acid-engineered proteins to be detected were synthesized in a residue-specific manner using a selective pressure incorporation method, and purified by metal affinity chromatography, semi-quantitative evaluation was performed.
A semi-quantitative assessment of the degree of non-protein amino acid substitution in macromolecular proteins has been achieved, breaking through the technical bottleneck of the assessment of macromolecular proteins by traditional methods.
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Figure CN119715853A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological detection, in particular to a semi-quantitative evaluation method for the degree of non-protein amino acid substitution. Background Art
[0002] Non-protein amino acids refer to compounds containing amino and carboxyl groups other than the 22 standard amino acids that make up natural proteins. Compared with standard amino acids, non-protein amino acids have the advantage of structural diversity, so they have become an important means to improve protein structure and performance. At present, more than 1,000 non-protein amino acids have been found in nature, of which more than 200 non-protein amino acids are used for engineering protein modification.
[0003] Evaluation of the specific position and degree of non-protein amino acid substitution for its parent amino acid is an important prerequisite for exploring its influence on protein structure and function. According to relative molecular weight, proteins can be divided into two categories: small molecules (generally with a relative molecular weight of less than 25 kDa) and large molecules (generally with a relative molecular weight of more than 100 kDa); according to the method of modification, non-protein amino acid engineered proteins can be divided into two categories, site-specific incorporation and residue-specific incorporation. Therefore, non-protein amino acid engineered proteins can be roughly divided into four categories: the first category is small molecule proteins engineered by site-specific incorporation, the second category is large molecule proteins engineered by site-specific incorporation, the third category is small molecule proteins engineered by residue-specific incorporation, and the fourth category is large molecule proteins engineered by residue-specific incorporation. Among them, the first three categories can use modern science and technology to quantitatively or semi-quantitatively determine the position and degree of non-protein amino acid substitution for its parent amino acid. For the fourth category, modern science and technology can evaluate the substitution site of non-protein amino acids, but there is no feasible method to evaluate the degree of substitution of non-protein amino acids. Summary of the invention
[0004] The present invention is intended to provide a semi-quantitative assessment method for the degree of non-protein amino acid substitution to solve the problem.
[0005] The semi-quantitative evaluation method of the degree of non-protein amino acid substitution in this scheme comprises the following steps: Step S1: expressing a recombinant plasmid and synthesizing a non-protein amino acid engineered protein to be tested: using phenylalanine-deficient E. coli cells, synthesizing a non-protein amino acid engineered protein to be tested in a residue-specific manner by a selective pressure incorporation method; the synthesized protein to be tested contains a histidine tag;
[0006] Step S2: Purifying the engineered protein to be detected: collecting host cells by centrifugation, lysing, and purifying by metal affinity chromatography to obtain a high-purity protein to be detected;
[0007] Step S3: preparing LC / MS-MS samples: washing the purified engineered protein to be detected, concentrating, drying, and hydrolyzing to obtain an aqueous solution of peptide fragments of the protein to be detected; purifying and concentrating by microchromatographic column to obtain LC / MS-MS samples;
[0008] Step S4: LC / MS-MS semi-quantitative evaluation of the degree of non-protein amino acid substitution: obtaining LC / MS-MS mass spectrometry data; selecting reference peptides, analyzing the mass spectrometry data, and achieving semi-quantitative evaluation of the degree of non-protein amino acid substitution.
[0009] Furthermore, the protein to be detected is a recombinant protein derived from prokaryotic cells or eukaryotic cells or viruses or archaea and located in the cytoplasm or cell membrane; the relative molecular weight of the wild-type protein is greater than 120 kDa; the number of phenylalanine residues in the amino acid sequence of the wild-type protein is greater than 40; in the present invention, the recombinant protein with a histidine tag is a peptide segment with multiple histidines fused to the end of the recombinant protein.
[0010] Furthermore, the non-protein amino acid engineered protein to be detected is prepared in a residue-specific manner by a selective pressure incorporation method; the selective pressure incorporation method refers to adding exogenous non-protein amino acids to an amino acid-deficient culture medium, culturing amino acid-deficient microbial cells, inducing the expression of target genes, and synthesizing the non-protein amino acid engineered protein to be detected; the residue-specific manner refers to the non-protein amino acids replacing phenylalanine in the amino acid sequence of the wild-type protein to be detected in a random manner.
[0011] Furthermore, the non-protein amino acid is fluorine-substituted phenylalanine, which refers to monofluorophenylalanine, difluorophenylalanine, trifluorophenylalanine, tetrafluorophenylalanine or pentafluorophenylalanine in which the hydrogen atoms on the benzene ring of phenylalanine are substituted by one or more fluorine atoms.
[0012] Further, the protein purification method is metal affinity chromatography, and the purity of the protein to be detected is greater than 90%; the LC / MS-MS sample is obtained by washing, concentrating, drying and purifying the protein, hydrolyzing, microchromatographic column purification, and concentrating; the hydrolysis method is a chemical method or a biological method;
[0013] The reagent used in the chemical method is cyanogen bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole or o-iodobenzoic acid or hydroxylamine or 2-nitro-5-thiocyanatobenzoic acid, and the added amount is 1-150 times the target hydrolyzed peptide bond amount; the amount of the protein to be detected is 0.5-40 mg / mL; the hydrolysis reaction is stirred and incubated in an acidic environment at 10-50° C. for 5-72 hours;
[0014] The enzyme used in the biological method is endopeptidase or cysteine protease or chymotrypsin or trypsin or clostripain or enterokinase or pepsin or thermolysin or coagulation protease, and the amount of protease added is 1:1-1:50 (protease / protein to be detected, mol / mol); the amount of protein to be detected is 0.05-10 mg / mL; the hydrolysis reaction is stirred and incubated at 10-50° C. for 5-72 hr;
[0015] Furthermore, the hydrolyzed peptide aqueous solution is purified and concentrated using a microchromatographic column; the microchromatographic column is BeyoDesalt TM G-25 series desalting columns or Millipore ZipTip series desalting columns or NW Dex G-25 series desalting columns or HisTRAP desalting columns.
[0016] Furthermore, the degree of substitution of non-protein amino acids in the protein to be detected is obtained by the degree of substitution of non-protein amino acids in a certain reference peptide segment; the selection criteria of the reference peptide segment are: (1) the peptide segment contains at least one phenylalanine residue; (2) at least one phenylalanine residue in the peptide segment is replaced by a non-protein amino acid; (3) the peptide segment does not contain an enzyme cleavage site; (4) the content of the peptide segment in different batches of hydrolyzed samples of the same protein is relatively stable, and the content of the peptide segment in the same batch and different batches of hydrolyzed samples of different proteins is relatively stable; (5) the peptide segment does not undergo intramolecular chemical reactions during the ionization process of the mass spectrometer; (6) the peptide segment does not undergo intermolecular chemical reactions during the ionization process of the mass spectrometer.
[0017] The working principle and beneficial effects of the present scheme are as follows: (1) The present invention can quickly and easily assess the degree of substitution of non-protein amino acids in proteins; (2) The present invention is not limited by the source of proteins and can be widely applied to proteins from various organisms and various locations in cells; (3) The present invention can identify isotope peaks, and the requirement for the difference in relative molecular weight between non-protein amino acids and their parent amino acids is at the isotope level, and is therefore basically not limited by the chemical structure of non-protein amino acids; (4) The present invention is not limited by the properties of non-protein amino acids, and does not require them to have spectral properties and post-translational modifications (such as fluorescent labels), etc., and can therefore be applied to most non-protein amino acids; (5) The present invention is not limited by the relative molecular weight of the wild-type protein to be detected, and can be applied to proteins with a relative molecular weight greater than 120 kDa, breaking through the technical bottleneck of assessing the degree of substitution of non-protein amino acids in macromolecular proteins and filling the gap in research technology in the field of protein engineering. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the amino acid chemical structure of the present invention; wherein a: L-phenylalanine; b: fluorine-substituted phenylalanine (L-type);
[0019] Figure 2 is the mass spectrum of the reference peptide; a: the mass spectrum of the wild-type peptide; b: the mass spectrum of the fluorine-substituted phenylalanine engineered peptide. DETAILED DESCRIPTION
[0020] The following is a further detailed description through specific implementation methods:
[0021] Reference Figure 1-2 A semi-quantitative evaluation method for the degree of non-protein amino acid substitution in a protein with a relative molecular weight greater than 120 kDa comprises the following steps:
[0022] Step S1: Expression of recombinant plasmid, synthesis of non-protein amino acid engineered protein to be tested
[0023] Using phenylalanine-deficient E. coli cells, a non-protein amino acid engineered protein to be tested is synthesized in a residue-specific manner by a selective pressure incorporation method; the synthesized protein to be tested contains a histidine tag.
[0024] The wild-type protein to be detected prepared by the present invention is a recombinant protein derived from prokaryotic cells or eukaryotic cells or viruses or archaea and located in the cytoplasm or cell membrane; the relative molecular weight of the wild-type protein is greater than 120kDa; the number of phenylalanine residues in the amino acid sequence of the wild-type protein is greater than 40; in the present invention, the recombinant protein is a histidine-tagged protein.
[0025] The protein to be detected containing non-protein amino acids prepared by the present invention is prepared in a residue-specific manner by a selective pressure incorporation method; the selective pressure incorporation method refers to adding exogenous non-protein amino acids to an amino acid-deficient culture medium, culturing amino acid-deficient microbial cells, inducing the expression of target genes, and synthesizing the protein to be detected engineered with non-protein amino acids; the residue-specific manner refers to the non-protein amino acids replacing phenylalanine in the amino acid sequence of the wild-type protein to be detected in a random manner; the present invention uses the selective pressure incorporation method to prepare the protein to be detected containing non-protein amino acids in a residue-specific manner, which is a method of operation well known to ordinary technicians in the field.
[0026] The non-protein amino acid used in the present invention is fluorine-substituted phenylalanine, which refers to monofluorophenylalanine, difluorophenylalanine, trifluorophenylalanine, tetrafluorophenylalanine or pentafluorophenylalanine in which the hydrogen atoms on the benzene ring of phenylalanine are replaced by one or more fluorine atoms, such as Figure 1 shown.
[0027] Step S2: Purification of engineered protein to be detected
[0028] The protein purification method used in the present invention is metal affinity chromatography, and the purity of the protein to be detected is greater than 90%.
[0029] Step S3: Preparation of LC / MS-MS samples
[0030] The invention obtains an LC / MS-MS sample by washing, concentrating, drying, and hydrolyzing the engineered protein to be detected, and then purifying and concentrating the protein through a microchromatographic column.
[0031] The hydrolysis method used in the present invention is a chemical method or a biological method; the reagent used in the chemical method is cyanogen bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole or o-iodoxybenzoic acid or hydroxylamine or 2-nitro-5-thiocyanatobenzoic acid, preferably cyanogen bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole or o-iodoxybenzoic acid; the added amount is 1-150 times the target hydrolyzed peptide bond amount, preferably 30-120 times; the amount of the protein to be detected is 0.5-40 mg / mL, preferably 1-20 mg / mL; the hydrolysis reaction is stirred and incubated in an acidic environment at 10-50° C. for 5-72 hours;
[0032] The protease used in the biological method is endopeptidase or cysteine protease or chymotrypsin or trypsin or clostripain or enterokinase or pepsin or thermophilic protease or coagulation protease, preferably cysteine protease or chymotrypsin or trypsin or clostripain or enterokinase or pepsin; the amount of protease added is 1:1-1:50 (protease / protein to be detected, mol / mol), and the optimal amount is 1:35; the amount of protein to be detected is 0.05-10 mg / mL, and the optimal amount is 0.1-5 mg / mL; the hydrolysis reaction is stirred and incubated at 10-50°C for 5-72 hr.
[0033] The peptide aqueous solution referred to in the present invention is purified and concentrated by a microchromatographic column to obtain a sample to be tested; the microchromatographic column used is BeyoDesalt TM G-25 series desalting column or Millipore ZipTip series desalting column or NW Dex G-25 series desalting column or HisTRAP desalting column, among which Millipore ZipTip series desalting column has the best effect.
[0034] Step S4: Semi-quantitative evaluation of non-protein amino acid substitution by LC / MS-MS
[0035] (1) LC / MS-MS experiments: The chromatographic columns were purchased from Dr. Maisch GmbH, Ammerbuch, Germany; the flow rate was 300 nL / min; the samples were eluted into the mass spectrometer with 2-90% mobile phase B within 60 min; mobile phase A consisted of water and 0.1% formic acid, and mobile phase B consisted of acetonitrile and 0.1% formic acid; the raw data of LC / MS-MS were processed with pFind (version 3.2.0) software, and the false discovery rate (FDR) of the mass spectrometer was <0.01 at the peptide and protein levels; LC / MS-MS testing was performed by the Public Technology Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
[0036] (2) Data analysis: The present invention obtains the degree of substitution of non-protein amino acids in the protein to be detected by analyzing the degree of substitution of non-protein amino acids in a certain reference peptide segment in the protein to be detected. The selection criteria of the reference peptide segment referred to in the present invention are: 1) the peptide segment contains at least one phenylalanine residue; 2) at least one phenylalanine residue in the peptide segment is replaced by a non-protein amino acid; 3) the peptide segment does not contain an enzyme cleavage site; 4) the content of the peptide segment is relatively stable in different batches of hydrolyzed samples of the same protein, and is relatively stable in the same batch and different batches of hydrolyzed samples of different proteins; 5) the peptide segment does not undergo intramolecular chemical reactions during the ionization process of the mass spectrometer; 6) the peptide segment does not undergo intermolecular chemical reactions during the ionization process of the mass spectrometer.
[0037] Embodiment 1:
[0038] See also Figure 1 to Figure 2 The present invention provides a technical solution: qualitative detection of a wild-type protein with a relative molecular weight of about 120.04 kDa
[0039] In a complete liquid medium, the gene of the protein to be detected in the phenylalanine-deficient E. coli cells is induced to express, and the wild-type protein to be detected is synthesized. The E. coli host cells are collected by centrifugation, lysed, and the protein to be detected is purified by metal affinity chromatography to obtain a 90% pure wild-type protein to be detected. Wash, concentrate, dry, and hydrolyze to obtain an aqueous peptide fragment of the wild-type protein to be detected. Purify and concentrate with a microchromatographic column to obtain an LC / MS-MS sample of the wild-type protein to be detected. Collect LC / MS-MS data and process the data. The results obtained are as follows:
[0040] (1) A total of 4 missing peptide segments were found, and none of the missing peptide segments contained phenylalanine residues. The information of the 4 missing peptide segments is shown in Table 1.
[0041] Table 1 Peptides missing from the mass spectrometry detection of wild-type protein in Example 1 of the present invention
[0042] Entry Amino acid residue position Number of missing residues Whether it contains phenylalanine residues 1 423-427 5 no 2 472-480 9 no 3 763-769 7 no 4 1011-1041 31 no
[0043] (2) The selected peptide contains two phenylalanine residues, and its mass spectrum is as follows Figure 2 As shown, it has a +4 charge.
[0044] Embodiment 2:
[0045] See also Figure 1 to Figure 2 The present invention provides a technical solution: semi-quantitative evaluation of non-protein amino acids in an engineered protein to be tested with a relative molecular weight greater than 120 kDa,
[0046] The parent protein corresponding to the protein engineered with non-protein amino acids is the wild-type protein in Example 1. In a phenylalanine-deficient liquid culture medium, fluorine-substituted phenylalanine is added to induce the expression of the protein gene to be detected in the phenylalanine-deficient E. coli cells to synthesize the engineered protein to be detected. The E. coli host cells are collected by centrifugation, lysed, and purified by metal affinity chromatography to obtain an engineered protein to be detected with a purity of 90%. Wash, concentrate, dry, and hydrolyze to obtain an aqueous solution of peptide segments of the engineered protein to be detected. Purify and concentrate with a microchromatographic column to obtain an LC / MS-MS sample of the engineered protein to be detected. Collect LC / MS-MS data and process the data. The results obtained are as follows:
[0047] (1) The selected peptide is the peptide in Example 1, and its mass spectrum is as follows Figure 2As shown, the peptide has a +4 charge.
[0048] (2) By Figure 2 The degree of substitution of non-protein amino acids in this peptide segment was calculated to be 78%, and thus it was concluded that the degree of substitution of non-protein amino acids in the engineered protein to be tested was 78%.
[0049] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A semi-quantitative evaluation method for the degree of non-protein amino acid substitution, characterized in that: The following steps are involved: Step S1: expressing the recombinant plasmid and synthesizing the non-protein amino acid engineered protein to be detected: using phenylalanine-deficient E. coli cells, synthesizing the non-protein amino acid engineered protein to be detected in a residue-specific manner through a selective pressure incorporation method; the synthesized protein to be detected contains a histidine tag; Step S2: Purifying the engineered protein to be detected: collecting host cells by centrifugation, lysing, and purifying by metal affinity chromatography to obtain a high-purity protein to be detected; Step S3: preparing LC / MS-MS samples: washing the purified engineered protein to be detected, concentrating, drying, and hydrolyzing to obtain an aqueous solution of peptide fragments of the protein to be detected; purifying and concentrating by microchromatographic column to obtain LC / MS-MS samples; Step S4: LC / MS-MS semi-quantitative evaluation of the degree of non-protein amino acid substitution: obtaining LC / MS-MS mass spectrometry data; selecting reference peptides, analyzing the mass spectrometry data, and achieving semi-quantitative evaluation of the degree of non-protein amino acid substitution.
2. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 1, characterized in that: The protein to be detected is a recombinant protein derived from prokaryotic cells, eukaryotic cells, viruses or archaea and located in the cytoplasm or cell membrane; the relative molecular weight of the wild-type protein is greater than 120kDa; the number of phenylalanine residues in the amino acid sequence of the wild-type protein is greater than 40; in the present invention, the recombinant protein with a histidine tag is a peptide segment with multiple histidines fused to the end of the recombinant protein.
3. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 2, characterized in that: The non-protein amino acid engineered protein to be detected is prepared in a residue-specific manner by a selective pressure incorporation method; the selective pressure incorporation method refers to adding exogenous non-protein amino acids to an amino acid-deficient culture medium, culturing amino acid-deficient microbial cells, inducing the expression of target genes, and synthesizing the non-protein amino acid engineered protein to be detected; the residue-specific manner refers to the non-protein amino acids replacing phenylalanine in the amino acid sequence of the wild-type protein to be detected in a random manner.
4. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 3, characterized in that: The non-protein amino acid is fluorine-substituted phenylalanine, which refers to monofluorophenylalanine, difluorophenylalanine, trifluorophenylalanine, tetrafluorophenylalanine or pentafluorophenylalanine in which the hydrogen atoms on the benzene ring of phenylalanine are replaced by one or more fluorine atoms.
5. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 4, characterized in that: The sample form of the protein to be detected is an aqueous solution of peptide fragments after chemical or biological hydrolysis; The reagent used in the chemical method is cyanogen bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole or o-iodobenzoic acid or hydroxylamine or 2-nitro-5-thiocyanatobenzoic acid, and the added amount is 1-150 times the target hydrolyzed peptide bond amount; the amount of the protein to be detected is 0.5-40 mg / mL; the hydrolysis reaction is stirred and incubated in an acidic environment at 10-50° C. for 5-72 hours; The enzyme used in the biological method is endopeptidase or cysteine protease or chymosin or trypsin or clostripain or enterokinase or pepsin or thermolysin or coagulation protease, and the amount of protease added is 1:50-1:1 (protease / protein to be detected, mol / mol); the amount of protein to be detected is 0.05-10 mg / mL; the hydrolysis reaction is stirred and incubated at 10-50° C. for 5-72 hr.
6. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 5, characterized in that: The hydrolyzed peptide aqueous solution is purified and concentrated using a microchromatographic column; the microchromatographic column is BeyoDesalt TM G-25 series desalting columns or Millipore ZipTip series desalting columns or NW Dex G-25 series desalting columns or HisTRAP desalting columns.
7. The semi-quantitative evaluation method for the degree of non-protein amino acid substitution according to claim 6, characterized in that: The degree of substitution of non-protein amino acids in the protein to be detected is obtained by the degree of substitution of non-protein amino acids in a certain reference peptide segment; the selection criteria of the reference peptide segment are: (1) the peptide segment contains at least one phenylalanine residue; (2) at least one phenylalanine residue in the peptide segment is replaced by a non-protein amino acid; (3) the peptide segment does not contain an enzyme cleavage site; (4) the content of the peptide segment in different batches of hydrolyzed samples of the same protein is relatively stable, and the content of the peptide segment in the same batch and different batches of hydrolyzed samples of different proteins is relatively stable; (5) the peptide segment does not undergo intramolecular chemical reactions during the ionization process of the mass spectrometer; (6) the peptide segment does not undergo intermolecular chemical reactions during the ionization process of the mass spectrometer.