CN113933420A - Mass spectrum-based monoclonal antibody head-on sequencing method - Google Patents
Mass spectrum-based monoclonal antibody head-on sequencing method Download PDFInfo
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Abstract
The invention provides a mass spectrum-based monoclonal antibody de-novo sequencing method, which belongs to the field of biological medicine characterization, high-quality monoclonal antibody multi-enzymolysis liquid quality data are obtained through high performance liquid chromatography separation and mass spectrum detection, a secondary spectrum is subjected to de-novo sequencing and sequence assembly through sequencing software to obtain an amino acid sequence of a monoclonal antibody, and after a sequence output by the software is subjected to molecular weight verification and manual further analysis, the light and heavy chain sequence of the monoclonal antibody can reach 100% coverage rate and 99.5% or more amino acid accuracy, so that a method is provided for accurate and complete de-novo sequencing of an unknown monoclonal antibody in sequence to ensure the biological activity of the monoclonal antibody.
Description
Technical Field
The invention belongs to the field of biological medicine characterization, and particularly relates to a mass spectrum-based monoclonal antibody head-on sequencing method.
Background
In recent years, with the advent of more and more antibody drugs, accurate sequencing and structural characterization of antibodies has become increasingly important in the drug development process. In particular, the sequence of the complementarity determining region, i.e., the CDR region, of an antibody is an important factor in the activity of an antibody.
De novo sequencing of monoclonal antibodies has been performed by both the Edman degradation method and mass spectrometry-based de novo sequencing. Edman degradation on monoclonalsThe N-terminus of the antibody was sequenced. Usually, the first 30 amino acids of the N-terminus are determined, and the full-length sequence of the monoclonal antibody is not obtained. The mass spectrometry-based de novo sequencing technique utilizes the precise mass number of primary parent ions and the abundance of multiple fragment ions (a, b, c, x, y, and b/y-H) in the secondary spectrum2O/NH3Etc.) to sequence the amino acids of the peptide segment. And then, carrying out sequence assembly by using the high-reliability peptide fragments obtained by sequencing, and simultaneously, assisting to search by using a homologous database to obtain the full-length sequence of the light and heavy chains of the monoclonal antibody. At present, neural networks and deep learning are added into the frontier de novo sequencing software to improve the sequencing accuracy, and in addition, open search of post-translational modification and scoring of amino acid reliability are supported.
However, mass spectrometry-based de novo sequencing techniques still face a number of challenges. For example, in distinguishing between isomeric leucines and isoleucine, the information in the primary mass spectrum and the secondary mass spectrum in the conventional dissociation mode (e.g., HCD) does not allow discrimination between the two amino acids. With the upgrade of mass spectra and the application of new dissociation modes, researchers found that fragmentation of peptide fragments containing I/L using EThcD resulted in mass differences of 29Da and 43Da, respectively, thereby achieving the discrimination of the two amino acids. However, not all peptide fragments containing I/L can obtain ideal EThcD spectrogram, so that three means are combined to distinguish I/L at present, namely the proportion of I/L in a same source database, enzyme cutting specificity (Chymotrypsin and Pepsin carry out enzyme cutting after L) and spectrogram information in EThcD.
Disclosure of Invention
Based on the problems in the prior art, the invention provides a mass spectrum-based monoclonal antibody de-novo sequencing method, which comprises the steps of obtaining high-quality monoclonal antibody multi-enzymatic hydrolysis liquid quality data through high performance liquid chromatography separation and mass spectrum detection, and obtaining an amino acid sequence of a monoclonal antibody through de-novo sequencing and sequence assembly of a secondary spectrum by sequencing software. After the sequence output by the software is subjected to molecular weight verification and manual further analysis, the light and heavy chain sequence of the monoclonal antibody can reach 100% coverage rate and more than 99.5% amino acid accuracy, so that a method is provided for accurate and complete de novo sequencing of the monoclonal antibody with unknown sequence to ensure the biological activity of the monoclonal antibody.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for sequencing a monoclonal antibody from head based on mass spectrum comprises the steps of preparing a monoclonal antibody sample into polypeptide through denaturation reduction, alkylation, desalting and enzymolysis in sequence, obtaining high-quality monoclonal antibody multiple enzymolysis liquid mass data through high performance liquid chromatography separation and mass spectrum detection, and obtaining an amino acid sequence of the monoclonal antibody through sequencing from head and sequence assembly of a secondary spectrum through sequencing software.
According to the above scheme, the method for sequencing the monoclonal antibody based on the mass spectrum from the head comprises the following detailed steps:
step S1, adding 6M guanidine hydrochloride buffer solution and 1M Dithiothreitol (DTT) into the monoclonal antibody sample, and reacting at 56 ℃ for 30min for denaturation reduction;
step S2, cooling the denatured and reduced monoclonal antibody sample to room temperature, adding 1M Iodoacetamide (IAA), and reacting for 30min at room temperature in a dark place for alkylation;
step S3, desalting the monoclonal antibody sample after alkylation using a zeba desalting column;
step S4, adding Trypsin (Trypsin) into the desalted monoclonal antibody sample, performing enzymolysis for 10-18 hours at room temperature, adjusting the pH value of the solution to acidity, and stopping enzymolysis to obtain a polypeptide sample;
step S5, performing reversed phase chromatography on the polypeptide sample obtained in the step S4 to separate a peptide fragment, performing Mass spectrum detection, analyzing the obtained original Data by using commercial de novo sequencing software Peaks AB, and selecting Correct precursor-Mass only by Data refine; selecting specific by each sample in an enzymolysis mode; fixed modification selection Carbamimetylation (+57.0215 Da); the maximum number of variable modifications allowed per peptide was 3; and performing de novo sequencing on the peptide fragment by using the accurate mass number of the primary parent ion and the abundant multiple fragment ions in the secondary spectrogram, and then re-assembling the peptide fragment sequence into a monoclonal antibody sequence.
According to the above scheme, the amount of Dithiothreitol (DTT) added in step S1 is added to a final concentration of 10 mM; in step S2 Iodoacetamide (IAA) was added in an amount to give a final concentration of 20 mM.
According to the above scheme, the step S4 further includes a step S41, in which the content of monoclonal antibody in the sample solution is determined, and the mass ratio of monoclonal antibody: trypsin 50: 1 to the monoclonal antibody sample solution trypsin was added.
According to the above scheme, the chromatographic conditions are as follows, the chromatographic column is C18, the mobile phase A is 0.1% FA aqueous solution, the mobile phase B is 0.1% FA acetonitrile solution, the flow rate is 0.3mL/min, and the gradient elution is carried out:
the column temperature was 55 deg.C, the sample chamber temperature was 5 deg.C, and the loading volume was 2. mu.L.
According to the scheme, the mass spectrum is under the following conditions that the spray voltage is 3.8kV, the collection is carried out in a positive ion mode, the temperature of a capillary tube is 320 ℃, and the S-lens RF level is 50; parent ion scan range: 300-2000 m/z; secondary mass spectrometry data was selected for dissociation of the first ten parent ions in intensity in dependent mode (DDA) with a Normalized Collision Energy (NCE) of 27%.
The invention has the beneficial effects that:
1. compared with the existing method for searching and identifying the protein by the database, the method provided by the invention has the advantage of finding new protein with unknown sequence.
2. Mass spectrometry-based de novo sequencing techniques can provide full-length sequences of monoclonal antibodies, compared to conventional Edman sequencing of no more than 30 amino acids at the N-terminus.
3. Further validation of molecular weight and manual secondary confirmation of amino acids in the light and heavy chain sequences of monoclonal antibodies ensure high-confidence sequence export.
Drawings
FIG. 1 is a main flow chart of a mass spectrometry-based monoclonal antibody de novo sequencing method.
FIG. 2 is a graph of the coverage of the monoclonal antibody sequences obtained by software sequencing assembly in the examples.
FIG. 3 is a graph showing the molecular weight verification results of the monoclonal antibodies of the examples.
FIG. 4 is a de novo sequencing secondary spectrum of the example.
FIG. 5 is a graph showing the results of combining the three discrimination means for leucine (L) and isoleucine (I).
Fig. 2 to 5 are diagrams of detection and analysis results, which are results shown in the examples, and the characters in the diagrams are results shown, and will change according to the result of each detection and analysis, that is, the characters in the diagrams are irrelevant to whether the detection method provided by the present invention can be repeatedly implemented, and the characters in the diagrams are unclear, so that the detection method provided by the present invention can be repeatedly implemented by those skilled in the art is not affected.
Detailed Description
The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.
Iodoacetamide (IAA) and guanidine hydrochloride (GdnHCl) used in this example were purchased from Sigma-Aldrich; dithiothreitol (DTT), Tris (hydroxymethyl) aminomethane (Tris) were purchased from Bio-Rad; trypsin (Trypsin) was purchased from Promega; the desalting column zeba was purchased from Thermo Fisher; water (H)2O) and Acetonitrile (ACN) were purchased from Fisher scientific; formic Acid (FA) was purchased from Fluka.
The method for sequencing the monoclonal antibody from the head based on the mass spectrum as shown in figure 1 comprises the following steps of firstly preparing a monoclonal antibody sample into polypeptide through steps of denaturation reduction, alkylation, desalting and enzymolysis in sequence, then obtaining high-quality monoclonal antibody multi-enzymolysis liquid quality data through high performance liquid chromatography separation and mass spectrum detection, and obtaining an amino acid sequence of the monoclonal antibody through de-head sequencing and sequence assembly of a secondary spectrum by sequencing software, wherein the method comprises the following detailed steps:
step S1, adding 6M guanidine hydrochloride buffer solution into 100 μ g of monoclonal antibody sample, adding 1M dithiothreitol until the final concentration of dithiothreitol is 10mM, and reacting at 56 deg.C for 30min for denaturation reduction.
And step S2, cooling the denatured and reduced monoclonal antibody sample to room temperature, adding 1M iodoacetamide until the final concentration of the iodoacetamide is 20mM, and reacting for 30min at room temperature in a dark place for alkylation.
At step S3, the monoclonal antibody samples after alkylation were desalted using a zeba desalting column according to the manufacturer' S operating manual.
Step S4, taking the monoclonal antibody sample after desalting, determining the content of the monoclonal antibody in the sample solution, and then according to the mass ratio of the monoclonal antibody: trypsin 50: 1, adding trypsin into the monoclonal antibody sample solution, carrying out enzymolysis for 12 hours at room temperature, adjusting the pH of the solution to acidity, stopping enzymolysis, obtaining a polypeptide sample after the enzymolysis of the monoclonal antibody, carrying out concentration measurement, then carrying out split charging, and preserving the temporarily unused polypeptide sample at-80 ℃.
Step S5, the polypeptide sample obtained in the step S4 is taken to use reversed phase chromatography to separate peptide fragments, then a high resolution orbital hydrazine mass spectrometer is used for detection,
the chromatographic conditions were as follows: the chromatographic column is C18, the mobile phase A is 0.1% FA aqueous solution, the mobile phase B is 0.1% FA acetonitrile solution, the flow rate is 0.3mL/min, and the gradient elution is carried out:
the column temperature was 55 deg.C, the sample chamber temperature was 5 deg.C, and the loading volume was 2. mu.L.
The mass spectrometry conditions were as follows: the spray voltage is 3.8kV, the collection is carried out in a positive ion mode, the temperature of a capillary tube is 320 ℃, and the S-lens RF level is 50 ℃; parent ion scan range: 300-2000 m/z; secondary mass spectrometry data was selected for dissociation of the first ten parent ions in intensity in dependent mode (DDA) with a Normalized Collision Energy (NCE) of 27%.
The raw data obtained were analyzed using commercial de novo sequencing software peak AB. Data refine selects Correct precursor-Mass only; selecting specific by each sample in an enzymolysis mode; fixed decoration is selectedCarbammidomethylation (+57.0215 Da); the maximum number of variable modifications allowed per peptide was 3. De novo sequencing of monoclonal antibodies involves two aspects (FIG. 1), one is the use of the exact mass number of the primary parent ion and the abundance of various fragment ions (a, b, c, x, y, and b/y-H) in the secondary spectrum2O/NH3Etc.) and on the one hand, the assembly of the peptide fragments into the monoclonal antibody sequences. After software analysis, the information of the amino acid sequence, the coverage rate and the molecular weight of the monoclonal antibody light and heavy chains can be obtained.
In the monoclonal antibody sequence coverage map (FIG. 2) obtained after software sequencing assembly, each amino acid in the sequence is reliably sequenced, and the de novo score is more than 85 percent. In general, the coverage rate of the monoclonal antibody light and heavy chain sequences reaches 100%.
And after the light chain and the heavy chain amino acid sequences of the monoclonal antibodies are obtained by software sequencing and assembling, verifying the molecular weight of the light chain and the heavy chain of the monoclonal antibodies. The experimental and theoretical molecular weights of the mabs matched, and the results further indicated the accuracy of the de novo sequencing technique based on mass spectrometry mabs (fig. 3).
There are many factors that affect the accuracy of the de novo sequencing results. For amino acids or amino acid combinations with very similar masses, a mass spectrometer with higher mass precision is selected for sequencing, so that a more accurate sequencing result is obtained. In addition, factors such as partial fragment ion deletion, misclassification of b and y ions, interference from other types of fragment ions, co-elution peptide fragment mixing profiles, post-translational modifications (e.g., N-glycosylation), and noise peaks can all reduce the accuracy of sequencing results. Therefore, the quality of the chromatographic separation is also of great importance for de novo sequencing of the monoclonal antibodies. FIG. 4 is an example of a de novo sequencing secondary profile; the peptide segment contains 31 amino acids, the molecular weight is 3663.7844, the score of-10 lgP is 200.00, and the charge z is 4; for multi-charge peptide fragments with longer length, accurate sequencing can be obtained under the condition of better secondary spectrum dissociation.
The current distinction for isomeric leucines (L) and isoleucine (I) is mainly a pattern of a combination of three approaches (fig. 5). The first is homology database search, and the probability of the site being I or L is determined according to the probability that the amino acid in the homologous protein is I or L; the second method is enzyme cutting specificity, both Chymotrypsin and Pepsin can be cut after L, and the C end of the peptide segment obtained by enzymolysis of the two enzymes has higher probability of being L rather than I. The third method is that the mass difference between z ions and w ions generated after the I and L are dissociated by the EThcD is 29Da and 43Da respectively, and the I and L can be distinguished according to the corresponding mass difference in the secondary spectrum. The combined mode of the three means can accurately distinguish leucine from isoleucine.
The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (6)
1. A mass spectrum-based monoclonal antibody de-novo sequencing method is characterized in that a monoclonal antibody sample is sequentially subjected to denaturation reduction, alkylation, desalting and enzymolysis to prepare polypeptide, high-quality monoclonal antibody multi-enzymolysis liquid quality data is obtained by high performance liquid chromatography separation and mass spectrum detection, and a secondary spectrum is subjected to de-novo sequencing and sequence assembly by sequencing software to obtain an amino acid sequence of a monoclonal antibody.
2. The method for sequencing a monoclonal antibody from the head based on mass spectrum according to claim 1, characterized in that it comprises the following detailed steps:
step S1, adding 6M guanidine hydrochloride buffer solution and 1M dithiothreitol into the monoclonal antibody sample, and reacting at 56 ℃ for 30min for denaturation reduction;
step S2, cooling the denatured and reduced monoclonal antibody sample to room temperature, adding 1M iodoacetamide, and reacting for 30min at room temperature in a dark place for alkylation;
step S3, desalting the monoclonal antibody sample after alkylation using a zeba desalting column;
step S4, adding trypsin into the monoclonal antibody sample after desalting for enzymolysis for 10-18 hours at room temperature, and adjusting the pH of the solution to acidity to stop enzymolysis to obtain a polypeptide sample;
step S5, taking the polypeptide sample obtained in the step S4, using reversed phase chromatography to separate the peptide fragment, then performing Mass spectrum detection, analyzing the obtained original Data by using commercial de novo sequencing software Peaks AB, and selecting Correct precorsor-Mass only by Data refine; selecting specific by each sample in an enzymolysis mode; fixed modification selection Carbamimetylation (+57.0215 Da); the maximum number of variable modifications allowed per peptide was 3; and performing de novo sequencing on the peptide fragment by using the accurate mass number of the primary parent ions and various fragment ions abundant in the secondary spectrogram, and then re-assembling the peptide fragment sequence into a monoclonal antibody sequence.
3. The method of claim 2, wherein the dithiothreitol is added to a final concentration of 10mM in step S1; the amount of iodoacetamide added in step S2 was added to a final concentration of 20 mM.
4. The method of claim 3, wherein the step S4 further comprises a step S41, wherein the content of monoclonal antibodies in the sample solution is determined, and the mass ratios of the monoclonal antibodies: trypsin 50: 1 to the monoclonal antibody sample solution trypsin was added.
5. The method for sequencing a monoclonal antibody from the head based on mass spectrometry of claim 3, wherein the chromatographic conditions are as follows, the chromatographic column is C18, the mobile phase A is 0.1% FA aqueous solution, the mobile phase B is 0.1% FA acetonitrile solution, the flow rate is 0.3mL/min, and the gradient elution is carried out:
the column temperature was 55 deg.C, the sample chamber temperature was 5 deg.C, and the loading volume was 2. mu.L.
6. The method of claim 3, wherein the mass spectrometry conditions are that the spray voltage is 3.8kV, the collection is performed in positive ion mode, the capillary temperature is 320 ℃, and the S-lens RF level is 50; parent ion scan range: 300-2000 m/z; the second-order mass spectrometry selects the parent ions with the first ten intensities to be dissociated in a data-dependent mode, and the normalized collision energy is 27%.
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| CN106645456A (en) * | 2016-10-13 | 2017-05-10 | 上海市食品药品检验所 | Mass-spectrum-technology-based sequencing method for monoclonal antibody medicine amino acid sequence |
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| US20190083591A1 (en) * | 2016-03-16 | 2019-03-21 | Immunocore Limited | Peptides |
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| US20190083591A1 (en) * | 2016-03-16 | 2019-03-21 | Immunocore Limited | Peptides |
| CN106645456A (en) * | 2016-10-13 | 2017-05-10 | 上海市食品药品检验所 | Mass-spectrum-technology-based sequencing method for monoclonal antibody medicine amino acid sequence |
| CN109425662A (en) * | 2017-08-23 | 2019-03-05 | 深圳华大基因研究院 | A kind of method and system for identifying albumen |
Non-Patent Citations (3)
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| JING ZHANG ET AL: "PEAKS DB: De Novo Sequencing Assisted Database Search for Sensitive and Accurate Peptide Identification", MOLECULAR & CELLULAR PROTEOMICS, vol. 11, no. 4, pages 1 - 8 * |
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