阅读说明：本技术 一种蛋白质氨基酸序列覆盖度的检测方法 (Method for detecting coverage of protein amino acid sequence ) 是由 张文明 杨兵 韩继臣 于 2019-02-18 设计创作，主要内容包括：本发明提供了一种蛋白质氨基酸序列覆盖度的检测方法。该蛋白质氨基酸序列覆盖度的检测方法包括蛋白还原烷基化、蛋白酶解、多肽脱盐、液质检测和数据分析,其中,蛋白酶解是通过加入弹性蛋白酶进行单酶切获得肽段,弹性蛋白酶与待检测的蛋白质的质量比为1：(20~100)。本发明的蛋白质氨基酸序列覆盖度的检测方法采用弹性蛋白酶实现单酶切,不需要模拟酶切预测来选择对应的酶；不需要多种酶切,只要一种酶；同时采用HLB柱脱盐,不会漏掉亲水性多肽,提高覆盖度；本发明的检测方法的快速简便、工作量少、检测周期短、价格经济,能够达到100%的氨基酸序列覆盖度。(The invention provides a method for detecting the coverage of a protein amino acid sequence. The method for detecting the coverage of the protein amino acid sequence comprises protein reduction alkylation, proteolysis, polypeptide desalination, liquid quality detection and data analysis, wherein the proteolysis is to obtain a peptide segment by adding elastase to carry out single enzyme digestion, and the mass ratio of the elastase to the protein to be detected is 1: (20-100). According to the method for detecting the coverage of the protein amino acid sequence, the single enzyme digestion is realized by adopting the elastase, and the corresponding enzyme is selected without simulating enzyme digestion prediction; multiple enzyme digestion is not needed, and only one enzyme is needed; meanwhile, hydrophilic polypeptide cannot be leaked out by adopting the HLB column for desalination, so that the coverage is improved; the detection method of the invention has the advantages of rapidness, simplicity, convenience, less workload, short detection period and economic price, and can reach 100 percent of amino acid sequence coverage.)

1. A detection method of protein amino acid sequence coverage degree comprises protein reduction alkylation, proteolysis, polypeptide desalination, liquid quality detection and data analysis, and is characterized in that:

the proteolysis is to obtain a peptide fragment by adding elastase to carry out single enzyme digestion, wherein the mass ratio of the elastase to the protein to be detected is 1: (20-100).

2. The detection method according to claim 1, wherein the polypeptide desalting is desalting treatment of the peptide segment after proteolysis, and specifically comprises:

firstly, adding trifluoroacetic acid into a peptide fragment, loading, slowly passing through an HLB column once, and ensuring that a sample is fully combined with the column; then adding trifluoroacetic acid to wash the column, repeating for 2 times; and finally, eluting the peptide segment bound on the HLB column by using a mixed solution of trifluoroacetic acid, water and acetonitrile, repeating for 1 time, and combining the eluates.

3. The detection method according to claim 1, wherein the steps of adding elastase for single enzyme digestion to obtain the peptide fragment are as follows:

and centrifuging the reduced and alkylated protein sample, adding an elastase solution, uniformly mixing at constant temperature of 37 ℃ and 500rpm, and carrying out enzyme digestion for 15 min-2 h.

4. The detection method according to claim 3, wherein the preparation method of the elastase solution comprises: 1mg of elastase was weighed, resuspended in 0.5mL of double distilled water, and diluted to a final concentration of 1mg/mL after quantification of protein concentration by BCA.

5. The detection method according to claim 1, wherein the method for reductive alkylation of protein comprises acetone precipitation and dilution; the acetone precipitation method is suitable for a protein sample to be tested containing a detergent; the dilution method is applied to a protein sample to be tested which does not contain a detergent; the detergent includes NP40 and tween 80.

6. The detection method according to claim 5, wherein the acetone precipitation method comprises the following steps:

weighing a protein sample to be detected, adding sodium dodecyl sulfate, uniformly mixing, and then adding triethylammonium bicarbonate to adjust the pH to 8; adding tri (2-carboxyethyl) phosphine to the bottom of the mixture solution, and carrying out constant-temperature reduction reaction after instantaneous centrifugation; after the reduction reaction is finished, adding iodoacetamide for constant-temperature alkylation reaction, and after the alkylation reaction is finished, adding acetone to precipitate protein; after centrifugal washing, triethylammonium bicarbonate is added to fully dissolve protein precipitate.

7. The detection method according to claim 5, wherein the dilution method comprises the following specific steps of reductive alkylation of the protein to be detected:

weighing a protein sample to be detected, adding urea, uniformly mixing, and then adding triethylammonium bicarbonate to adjust the pH to 8; adding tri (2-carboxyethyl) phosphine to the bottom of the mixture solution, and carrying out constant-temperature reduction reaction after instantaneous centrifugation; after the reduction reaction is finished, adding iodoacetamide for constant-temperature alkylation reaction, and after the alkylation reaction is finished, adding triethylammonium bicarbonate to dilute the reaction product.

8. The detection method according to claim 1, wherein in the liquid chromatography-mass spectrometry used for the liquid quality detection, the detection parameters of the liquid chromatography are as follows:

using a C18 column, single column mode; phase A is 0.1% FA water, phase B is 0.1% FA acetonitrile; the flow rate of the nanoliter liquid phase is 0.3 microliter/min, the linear gradient is 2-20% B, and the time is 30 minutes; 20-40% of B for 10 minutes; 40-80% of B for 2 minutes; 80% B,5 min; 80-2% of B for 1 minute; 2% B, 20 min; the flow rate of a conventional liquid phase is 200 microliters/minute, the linear gradient is 2-20% B, and the time is 30 minutes; 20-40% of B for 10 minutes; 40-80% of B for 2 minutes; 80% B,5 min; 80-2% of B for 1 minute; 2% B, 20 min.

9. The detection method according to claim 1 or 8, characterized in that: when the liquid quality detection is carried out, in the adopted liquid chromatogram-mass spectrum, the detection parameters of the mass spectrum are as follows:

the primary scanning range is 200-2000, the resolution is larger than 30000, the secondary scanning range is 100-2000 or 4000, and the resolution is larger than 15000; in DDA mode, TOP10 or TOP20, charges are 1-6 or 2-6, and 10-15S is excluded after ion detection once.

10. The detection method according to claim 1, characterized in that: and the data analysis is to analyze the data of the liquid quality detection by adopting protein library searching software so as to judge the coverage of the amino acid sequence of the protein to be detected, wherein the protein library searching software comprises a protein discover and/or PEAKS.

11. The detection method according to claim 10, characterized in that the detection of the data analysis is set to: the first-level allowable error is 10-20 ppm; the second-level allowable error is 0.02-0.1 Da; setting enzyme cutting sites as non-specific; 5-50 amino acids in length; variable modification includes protein N-terminal acetylation and M oxidation; fixed modifications include C alkylation; the ions are b and y ions.

Technical Field

The invention belongs to the technical field of protein detection, and relates to a method for detecting the coverage of a protein amino acid sequence.

Background

At present, protein drugs, particularly antibody drugs, are the class of drugs with the fastest growth rate in the pharmaceutical field in recent years. The amino acid sequence is the primary structure of protein medicine and the basis of medicine research.

The sequencing method of protein mainly comprises an Edman degradation method and a mass spectrometry method. The Edman degradation method can measure 50-70 amino acids at the N terminal at most, so polypeptide drugs with shorter length can be sequenced by the Edman degradation method, but for protein drugs, the amino acid sequence usually reaches 100-1500, and the Edman degradation method is generally only used for measuring 15-30 amino acid sequences at the N terminal and is not suitable for protein full-sequence detection. If protein sequence detection is required, amino acid sequence coverage analysis is generally carried out by adopting a mass spectrum-based method, and the amino acid sequence coverage analysis is taken as an important component of physicochemical properties in the mass research of protein drugs.

The Edman degradation method adopts Phenyl Isothiocyanate (PITC) to react with N-terminal amino of the polypeptide and protein to be analyzed under alkaline conditions to generate a derivative of Phenylamino Thiocarbamide (PTC), and then the derivative is treated by acid, closed, and the N-terminal is selectively cut off to obtain the thiazolinone aniline derivative of the N-terminal amino acid residue. The derivative is then extracted with an organic solvent and under the action of an acid, a stable Phenylthiohydantoin (PTH) derivative is formed. By analyzing the produced thiohydantoin (PTH-amino acid) by HPLC, it is possible to identify which amino acid is. The polypeptide or protein with the N-terminal amino acid residue removed enters the next cycle and continues to be degraded.

Generally, the mass spectrometry uses Trypsin Trypsin to carry out enzyme digestion and desalination on a protein sample, the obtained peptide fragments enter mass spectrometry detection to obtain a primary spectrogram and a secondary spectrogram, the spectrograms are compared with a theoretical polypeptide sequence of the protein to obtain a polypeptide sequence of the sample, and then the polypeptide sequence detected by the sample is compared with the theoretical sequence of the protein, so that the amino acid sequence coverage of 60-90% of the theoretical sequence can be generally obtained.

In order to obtain all sequence information of the protein, generally, a plurality of proteases (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C and the like) are used for carrying out enzyme digestion on the protein respectively, so that the length of the polypeptide after enzyme digestion is between 5 and 50 amino acids, namely, the polypeptide has sequence specificity and simultaneously generates a better secondary mass spectrogram, and finally, the 100 percent coverage of the protein sequence is realized by splicing and analyzing peptide segments generated by different enzyme digestions.

However, several enzymes other than Trypsin, such as Chymotrypsin and Asp-N, Glu-C, Lys-C, are expensive. Different reaction systems are needed for different enzymes, and the operation is complicated. The reaction time is different and is different from 2 to 16 hours. Sometimes, even with multiple enzymes, 100% sequence coverage is not necessarily achieved.

Disclosure of Invention

Based on the problems of the prior art, the invention aims to provide a method for detecting the coverage of a protein amino acid sequence. According to the method, single enzyme digestion is realized by adopting the elastase, and the corresponding enzyme is selected without simulating enzyme digestion prediction; multiple enzyme digestion is not needed, and only one enzyme is needed; meanwhile, hydrophilic polypeptide cannot be leaked out by adopting the HLB column for desalination, so that the coverage is improved.

The purpose of the invention is realized by the following technical scheme:

the invention provides a detection method of protein amino acid sequence coverage, which comprises protein reduction alkylation, proteolysis, polypeptide desalination, liquid quality detection and data analysis, wherein the proteolysis is to obtain a peptide segment by adding elastase for single enzyme digestion, and the mass ratio of the elastase to the protein to be detected is 1: (20-100).

The elastase of the invention is commercially available from Promega under model V1891. The elastase is capable of cleaving preferentially at the C-terminus of alanine, valine, serine, glycine, leucine, or isoleucine.

In the above detection method, preferably, the polypeptide desalting is desalting treatment of a peptide segment after proteolysis, specifically:

firstly, adding trifluoroacetic acid into a peptide fragment, loading, slowly passing through an HLB column once, and ensuring that a sample is fully combined with the column; then adding trifluoroacetic acid to wash the column, repeating for 2 times; and finally, eluting the peptide segment bound on the HLB column by using a mixed solution of trifluoroacetic acid, water and acetonitrile, repeating for 1 time, and combining the eluates.

In the above detection method, preferably, the specific steps of adding elastase to perform single enzyme digestion to obtain the peptide fragment are as follows:

and centrifuging the reduced and alkylated protein sample, adding an elastase solution, and uniformly mixing and digesting at constant temperature of about 37 ℃ and 500rpm for 15 min-2 h.

In the above detection method, preferably, the method for preparing the elastase solution comprises: 1mg of elastase was weighed, resuspended in 0.5mL of double distilled water, and diluted to a final concentration of 1mg/mL after quantification of protein concentration by BCA.

In the above-mentioned detection method, preferably, the method for reductive alkylation of protein comprises acetone precipitation and dilution; the acetone precipitation method is suitable for a protein sample to be tested containing a detergent; the dilution method is applied to a protein sample to be tested which does not contain a detergent; the detergent comprises NP40, Tween 80 and the like.

In the above detection method, preferably, the acetone precipitation method comprises the following specific steps of:

weighing a protein sample to be detected, adding Sodium Dodecyl Sulfate (SDS), uniformly mixing, and then adding triethylammonium bicarbonate (TEAB) to adjust the pH to be 8; adding tri (2-carboxyethyl) phosphine (TCEP) to the bottom of the mixture solution, and carrying out constant-temperature reduction reaction after instantaneous centrifugation; after the reduction reaction is finished, adding iodoacetamide for constant-temperature alkylation reaction, and after the alkylation reaction is finished, adding acetone to precipitate protein; after centrifugal washing, triethylammonium bicarbonate (TEAB) was added to dissolve the protein precipitate sufficiently.

In the above detection method, preferably, the dilution method comprises the following specific steps of:

weighing a protein sample to be detected, adding urea, uniformly mixing, and then adding triethylammonium bicarbonate (TEAB) to adjust the pH to 8; adding tri (2-carboxyethyl) phosphine (TCEP) to the bottom of the mixture solution, and carrying out constant-temperature reduction reaction after instantaneous centrifugation; after the reduction reaction is finished, adding iodoacetamide for constant-temperature alkylation reaction, and after the alkylation reaction is finished, adding triethylammonium bicarbonate (TEAB) to dilute the reaction product.

In the above detection method, preferably, in the liquid chromatography-mass spectrometry used for the liquid quality detection, the detection parameters of the liquid chromatography are as follows:

using a C18 column, single column mode; phase A is 0.1% FA water, phase B is 0.1% FA acetonitrile; the flow rate of the nanoliter liquid phase is 0.3 microliter/min, the linear gradient is 2-20% B, and the time is 30 minutes; 20-40% of B for 10 minutes; 40-80% of B for 2 minutes; 80% B,5 min; 80-2% of B for 1 minute; 2% B, 20 min; the flow rate of a conventional liquid phase is 200 microliters/minute, the linear gradient is 2-20% B, and the time is 30 minutes; 20-40% of B for 10 minutes; 40-80% of B for 2 minutes; 80% B,5 min; 80-2% of B for 1 minute; 2% B, 20 min, but is not limited thereto.

In the above detection method, preferably, in the liquid chromatography-mass spectrometry used for the liquid quality detection, the detection parameters of the mass spectrometry are as follows:

the primary scanning range is 200-2000, the resolution is larger than 30000, the secondary scanning range is 100-2000 or 4000, and the resolution is larger than 15000; DDA mode, TOP10 or TOP20, charges 1-6 or 2-6, excluding 10-15S after one ion detection, but not limited thereto.

In the above detection method, preferably, the data analysis is to analyze the data of the liquid quality detection by using protein library searching software to determine the coverage of the amino acid sequence of the protein to be detected, wherein the protein library searching software includes, but is not limited to, a protein resolver and/or PEAKS.

In the above detection method, preferably, the detection of the data analysis is set as: the first-level allowable error is 10-20 ppm; the second-level allowable error is 0.02-0.1 Da; setting enzyme cutting sites as non-specific; 5-50 amino acids in length; variable modification includes protein N-terminal acetylation and M oxidation; fixed modifications include C alkylation; the ions are b and y ions.

Compared with a scheme of a multi-enzyme cleavage mass spectrometry, the method does not need to simulate enzyme cleavage prediction to select the corresponding enzyme; multiple enzyme digestion is not needed, and only one enzyme is needed, so that the method is more economical; a plurality of reaction systems are not needed, only one reaction system is needed, and manpower and materials are saved; the enzyme digestion is not needed to be carried out overnight, and generally only 1 hour is needed, so that the reaction time is saved; c18 SPE column is not used for desalination, and HLB column is used, so that hydrophilic polypeptide cannot be leaked, and coverage is improved; or some schemes are not desalted, but the desalting scheme interferes the detection of the hydrophilic peptide with peak in the same time period if the general samples are desalted because the salt exists in the samples. And simultaneously, the blockage of the liquid chromatographic column and the pollution of the mass spectrum can be reduced. The scheme has the advantages of more economical materials, less manual workload, shorter whole detection time and better coverage.

Compared with the Edman degradation method scheme, the method does not need to simulate enzyme digestion prediction to select the corresponding enzyme; multiple enzyme digestion is not needed, and only one enzyme is needed, so that the method is more economical; the method does not need to carry out HPLC separation on the sample for multiple times, does not need to collect the separated polypeptide peak, and has less sample requirement and less workload. And each polypeptide peak is subjected to Edman degradation sequencing, which is equivalent to multiple detections, and the method has the advantages of large workload, long period and high cost. When the N end of the protein is blocked, the N end peptide segment of the protein cannot be detected by an Edman degradation method or needs to be treated by adding enzyme; the invention is not limited by N-terminal modification, and has the advantages of less material and workload, short period and economic price.

The invention has the beneficial effects that:

according to the method for detecting the coverage of the protein amino acid sequence, the single enzyme digestion is realized by adopting the elastase, and the corresponding enzyme is selected without simulating enzyme digestion prediction; multiple enzyme digestion is not needed, and only one enzyme is needed; meanwhile, hydrophilic polypeptide cannot be leaked out by adopting the HLB column for desalination, so that the coverage is improved; the detection method is rapid, simple and convenient, has small workload, short detection period and economic price, generally only needs 15-60 minutes for enzyme digestion reaction time, and can reach 100% of amino acid sequence coverage.

Drawings

FIG. 1 is a schematic diagram of reductive alkylation, digestion and desalting processes of a protein sample to be tested in example 1 of the present invention;

FIG. 2 is a spectrum of mass spectrometric detection of a protein sample to be detected in example 1 of the present invention;

FIG. 3 is a spectrum of data analysis of a protein sample to be tested in example 1 of the present invention;

FIG. 4 is a dot diagram showing the amino acid sequence coverage and modification of the protein in the test protein sample in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

The chemical reagents and the like used in the following examples are commercially available unless otherwise specified.