CN111579702A

CN111579702A - Method for detecting coverage of protein amino acid sequence

Info

Publication number: CN111579702A
Application number: CN201910119275.XA
Authority: CN
Inventors: 张文明; 杨兵; 韩继臣
Original assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Current assignee: Shanghai Majorbio Bio Pharm Technology Co ltd
Priority date: 2019-02-18
Filing date: 2019-02-18
Publication date: 2020-08-25

Abstract

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.

Description

Method for detecting coverage of protein amino acid sequence

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.

Example 1

The embodiment provides a method for detecting the coverage of a protein amino acid sequence, which comprises the following steps:

step one, reducing and alkylating a protein sample to be detected (the protein sample adopted in the implementation is recombinant human growth hormone rhGH); the method specifically comprises the following steps:

the protein sample to be detected is reductively alkylated by the following method:

dilution (for protein samples without detergents like NP40 and Tween 80):

sampling: 100 mu g of protein sample to be detected is taken out and put into a 1.5mL Eppendorf centrifuge tube, 8M urea is added until the volume is 90 mu L, after Vortex mixing, 10 mu L of 1M TEAB is added, the pH value is adjusted to be about 8, and the total volume of the solution is 100 mu L. (the amount of protein to be measured is determined by the subsequent liquid phase, the amount of protein sample may be smaller in the case of a nanoliter liquid phase, the amount of protein sample may be larger in the case of a conventional liquid phase, and the final volume is adjusted according to the concentration of the original sample.)

Reduction: transferring 2 μ L of 0.5M TCEP (keeping the final concentration of TCEP at 10 mM) by a pipette, placing on a tube bottom, performing instantaneous centrifugation, placing on a constant temperature mixing instrument, incubating at about 37 deg.C and 500rpm for 1h, and observing that the protein solution is clear and has no flocculence.

Alkylation: after reduction, the sample is returned to room temperature, 2 muL of 1M iodoacetamide is added, the mixture is placed on a constant temperature mixing instrument after instantaneous centrifugation, and the mixture is incubated for 40min at about 25 ℃ and about 500rpm in the dark.

Diluting: after alkylation was complete, the samples were diluted 8-fold with 100mM TEAB.

And step two, adding elastase (the manufacturer is Promega, the model is V1891) to carry out single enzyme digestion to obtain a peptide fragment, wherein the mass ratio of the elastase to the protein to be detected is 1: (20-100); the method specifically comprises the following steps:

and (3) instantly centrifuging the reduced and alkylated protein sample (according to the ratio of enzyme to protein mass =1: 20-100), adding elastase into the sample, placing the sample on a constant-temperature mixing machine, and uniformly mixing and digesting the protein sample at about 37 ℃ and 500rpm for 15min to 1 hour. (1 mg of elastase was weighed and resuspended in 0.5mL of double distilled water, BCA was quantitated and diluted to a final concentration of 1mg/mL, stored at 4 ℃ for 2 weeks)

In specific examples such as: the 20KDa protein such as growth hormone can be digested for 30min at a ratio of 1: 20; the 65KDa protein such as HPV protein can be cut by enzyme at a ratio of 1:20 for 30min, taking out half, terminating with TFA, standing at 4 deg.C, reacting the rest half for 30min, and mixing the two samples; the 150kD antibody was cleaved at 1:20 for 1 hour.

Remarking: n-sugar-containing proteins such as antibodies require the use of PNGase F glycosidase to remove sugar chains.

Step three, desalting the peptide segment; the method specifically comprises the following steps:

loading: 200 μ L of 0.1% TFA solubilized the peptide fragment and was passed slowly over an HLB column (μ Elution HLB) once to ensure adequate binding of the sample to the column. The HLB column retains more polar polypeptides than the C18 column.

Cleaning: the column was washed with 200. mu.L of 0.1% TFA and repeated 2 times.

And (3) elution: the peptide bound to the column was eluted with 50. mu.L of 0.1% TFA, 70% ACN, repeated once, the eluates combined to a total volume of 100. mu.L, and concentrated to dryness in vacuo.

A schematic process diagram of the first step to the third step is shown in fig. 1.

Detecting the desalted peptide section by using a liquid chromatography-mass spectrum to obtain detection data; the method specifically comprises the following steps:

liquid chromatography: the use of a C18 column, single column format, ensured that the remaining hydrophilic peptide, which was not detected by the C18 column, was all detectable. Phase A is typically 0.1% FA water and phase B is typically 0.1% FA acetonitrile. The flow rate of the nanoliter liquid phase is generally 0.3 microliter/min, the linear gradient is 2-20% B, and the flow rate 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 generally 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. The A phase, B phase and gradient can be adjusted as appropriate.

Mass spectrum: 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.

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

And step five, carrying out data analysis on the detection data obtained in the step four by using protein library searching software to judge the coverage of the amino acid sequence of the protein to be detected. The method specifically comprises the following steps:

protein library searching software such as protein discover, PEAKS, etc. is used. 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 modifications such as protein N-terminal acetylation, M oxidation, etc.; fixed modifications such as C alkylation. The ions are b and y ions.

The experimental results are shown in table 1, fig. 3 and fig. 4, table 1 is a data table of the results of coverage of the test sequences of the protein samples to be tested in example 1 of the present invention:

table 1:

protein sample	-10lgP	Coverage degree	Polypeptide fragment	Average mass	PTM modification sites
						rhGH	682.94	100%	424	22129	C、O

As can be seen from table 1, the sequence coverage of the protein is 100%. As can be seen from one of the spectrograms of the data analysis of the protein sample to be detected in FIG. 3, fragment ions in the secondary spectrogram are well matched with b and y ions of a theoretical sequence, wherein the fragment ions matched with the y ions are shown in red and marked as yn, the fragment ions matched with the b ions are shown in blue and marked as bn, and the amino acid sequence in the protein to be detected is successfully identified by the secondary spectrogram. In conjunction with the protein amino acid sequence coverage and modification site map of FIG. 4, it can be seen from FIG. 4 that: bold letters indicate successful detection of the amino acid, orange background O above the letters indicates oxidation of the amino acid, red background C indicates alkylation of the amino acid, all bold letters indicate that all amino acids have been detected, and sequence coverage is 100%.

In the embodiment, the elastase is utilized to obtain polypeptides with proper length and complementary polypeptides of enough kinds through proper enzyme cutting sites and controlling conditions such as reaction concentration, reaction time and the like. And by materials that retain hydrophilic peptides, more complete polypeptides are recovered, achieving 100% sequence coverage of the protein.

The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to implement the same, and the present invention shall not be limited to the embodiments, i.e. the spirit of the present invention disclosed, and equivalent changes or modifications shall still fall within the scope of the present invention.

Claims

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:

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.