CN105277638A - Monoclonal antibody IgG mass spectrometry sequencing method - Google Patents
Monoclonal antibody IgG mass spectrometry sequencing method Download PDFInfo
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- CN105277638A CN105277638A CN201410350750.1A CN201410350750A CN105277638A CN 105277638 A CN105277638 A CN 105277638A CN 201410350750 A CN201410350750 A CN 201410350750A CN 105277638 A CN105277638 A CN 105277638A
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- monoclonal antibody
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- 238000012163 sequencing technique Methods 0.000 title abstract description 7
- 238000004949 mass spectrometry Methods 0.000 title abstract 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 36
- 229920001184 polypeptide Polymers 0.000 claims abstract description 29
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 22
- 230000029087 digestion Effects 0.000 claims abstract description 16
- 238000005932 reductive alkylation reaction Methods 0.000 claims abstract description 13
- 238000001962 electrophoresis Methods 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 238000001819 mass spectrum Methods 0.000 claims description 12
- 238000005259 measurement Methods 0.000 claims description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 8
- 229940088598 enzyme Drugs 0.000 description 13
- 102000013519 Lipocalin-2 Human genes 0.000 description 12
- 108010051335 Lipocalin-2 Proteins 0.000 description 12
- 238000012797 qualification Methods 0.000 description 10
- 239000000693 micelle Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108010019160 Pancreatin Proteins 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 229940055695 pancreatin Drugs 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 4
- 108090000284 Pepsin A Proteins 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 108091036078 conserved sequence Proteins 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000734 protein sequencing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 238000010844 nanoflow liquid chromatography Methods 0.000 description 1
- -1 potassium ferricyanide Chemical compound 0.000 description 1
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- 230000006798 recombination Effects 0.000 description 1
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- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a monoclonal antibody IgG mass spectrometry sequencing method including the following steps: carrying out reductive alkylation of a monoclonal antibody IgG; separating a heavy chain and a light chain of the reductive-alkylated monoclonal antibody IgG by an electrophoresis method; carrying out in-gel digestion of the heavy chain and the light chain of the monoclonal antibody IgG to obtain polypeptide fragments; adding the polypeptide fragments obtained from digestion into an LC-MS/MS mass spectrometer to obtain a fingerprint atlas; analyzing and finding out CDR1/2/3 sequences through the fingerprint atlas; and sequencing the CDR1/2/3 sequences.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of monoclonal antibody IgG mass spectrum sequence measurement.
Background technology
At present, mostly the preparation of monoclonal antibody IgG is to be prepared by hybridoma, so just need the hybridoma cell strain obtaining producing high specific and sensitivity by high-throughout screening, and produce high-quality monoclonal antibody IgG with this hybridoma cell strain.The high flux screening workload of hybridoma is huge, will ensure the stability of the hybridoma screened simultaneously, needs strict condition of storage.Therefore engineered means can be used, obtain the monoclonal antibody of high specific and sensitivity, this just needs the protein sequence being obtained high-quality monoclonal antibody IgG by order-checking, thus derives its gene order, whereby, the monoclonal antibody IgG of high-quality is prepared by the mode of genetic recombination.In view of mass-spectrometric technique is more and more extensive in the application in albumen field, the demand of monoclonal antibody technology of preparing development trend simultaneously, need a kind of new method efficiently measuring monoclonal antibody IgG sequence accurately, thus need to develop a kind of suitable monoclonal antibody sequencing technologies, for the preparation of high-quality monoclonal antibody IgG.
Summary of the invention
In order to solve above technical matters, the invention provides a kind of monoclonal antibody IgG mass spectrum sequence measurement, when the order-checking of monoclonal antibody IgG is reduced to CDR1/2/3 sequencing, monoclonal antibody IgG order-checking difficulty declines rapidly.
The present invention is achieved through the following technical solutions:
A kind of monoclonal antibody IgG mass spectrum sequence measurement, comprises the following steps:
(1) by monoclonal antibody IgG reductive alkylation;
(2) by electrophoresis method, the described monoclonal antibody IgG of reductive alkylation is separated heavy chain and light chain;
(3) heavy chain of in-gel digestion monoclonal antibody IgG and light chain obtain polypeptide fragment;
(4) enzyme is cut the polypeptide fragment obtained and is loaded onto LC-MS/MS instrument;
(5) CDR1/2/3 sequence is found out by NanoLC fingerprint map analyzing;
(6) CDR1/2/3 sequence.
In technique scheme, the reductive alkylation in described step (1) adopts DTT-IAM pattern.
In technique scheme, the electrophoresis method in described step (2) is conventional SDS-PAGE electrophoresis.
In technique scheme, the heavy chain of the in-gel digestion monoclonal antibody IgG in described step (3) and light chain adopt conventional in-geldigestion method.
In technique scheme, adding mass spectrometric enzyme in described step (4), to cut polypeptide sample size be 5-15 μ L.
The present invention is combined by LC-MS/MS method and receives upgrade liquid chromatogram method (NanoLC) order-checking of monoclonal antibody IgG is reduced to CDR1/2/3 sequencing, polypeptide spectrogram potential for CDR1/2/3 can be locked onto some region by the analysis of NanoLC finger-print, then these spectrograms of labor and obtain final CDR1/2/3 peptide sequence information, then the conserved sequence in monoclonal antibody IgG and final CDR1/2/3 peptide sequence are carried out splicing and can obtain complete monoclonal antibody IgG amino acid sequence, monoclonal antibody IgG order-checking difficulty is declined rapidly.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in detail.
Embodiment one
NGAL antibody mass spectrum checks order:
The order-checking of NGAL antibody mass spectrum comprises the following steps: 1, homemade NGAL monoclonal antibody (heavy chain amino acid sequence of antibody is shown in sequence table 1, and the light-chain amino acid sequence of antibody is shown in sequence table 2) is carried out reductive alkylation; 2, SDS-PAGE electrophoresis is through the NGAL monoclonal antibody IgG of reductive alkylation, is separated heavy chain and light chain; 3, the heavy chain of in-gel digestion NGAL monoclonal antibody IgG and light chain obtain polypeptide fragment respectively; 4, enzyme cuts LC-MS/MS instrument on the polypeptide that obtains; 5, nanoLC fingerprint figure analysis; 6, spectrogram qualification and CDR1/2/3 qualification.The operation of concrete each step is as follows:
1, NGAL monoclonal antibody reductive alkylation
Monoclonal antibody IgG reductive alkylation adopts DTT-IAM pattern, and DDT (Dithiothreitol, dithiothreitol (DTT)) is for Reduction of Disulfide, and IAM (iodacetyl ammonia) is for closed sulfydryl.The operation steps that the method is concrete is as follows: the IgG antibody solution (i.e. 20 μ g) 1. getting 20 μ L operates for reductive alkylation; 2. 0.2 μ LDTT is added, 56 DEG C of water-bath 45min; 3. ice bath 5min; Thoroughly cool to room temperature; 4. under dark condition, 2 μ LIAM are added, reaction 60min; 5. 13000rpm, centrifugal 5min, removes precipitation; 6. take out supernatant, supernatant is used for follow-up SDS-PAGE electrophoresis.
2, the heavy chain of SDS-PAGE electrophoretic separation antibody and light chain
SDS-PAGE electrophoresis is through the monoclonal antibody IgG of reductive alkylation, and be separated in heavy chain and light chain, SDS-PAGE electrophoresis adopts conventional SDS-PAGE electrophoresis method.
3, the heavy chain of in-gel digestion NGAL monoclonal antibody IgG and light chain
The heavy chain obtained electrophoretic separation by pancreatin, pepsin and elastoser respectively after electrophoretic separation completes and light chain carry out in-gel digestion, and its concrete enzyme cuts through journey and parameter is as follows:
Pancreatin in-gel digestion: adopt pancreatin to the heavy chain of monoclonal antibody IgG and light chain carry out in-gel digestion obtain polypeptide fragment time, in-gel digestion monoclonal antibody IgG adopts conventional in-geldigestion method.The operation steps that the method is concrete is as follows: 1. clean and decolour: protein spots bale cutting instrument is cut glue, is placed in 96 orifice plates.200/A1 destainer (100mmol/L sodium thiosulfate and 30mmol/L potassium ferricyanide solution mix according to 1:1) is added in containing each hole of micelle, after leaving standstill 30min, abandoning supernatant.Then add 200/ μ L water cleaning, after leaving standstill 30min, abandon supernatant.Repeat this step until micelle becomes colorless; 2. dehydrate: add after 200 μ L acetonitriles leave standstill 30min in micelle, abandon supernatant.Repeat this step to dewater completely to gel and bleach.After acetonitrile sucking-off being given up, micelle is placed in 37 DEG C to bone dry; 3. enzyme vacuole is swollen: after the trypsase 20mmol/L ammonium bicarbonate soln of order-checking level is formulated as concentration 12.5ng/mL, add appropriate enzyme liquid in micelle, and places 30min in 4 DEG C and micelle is steeped completely rise.Then the enzyme liquid that sucking-off is unnecessary discards, to prevent the peptide section occurring too much trypsase auto-degradation during Mass Spectrometer Method; 4. enzymolysis: the 25mmol/L ammonium bicarbonate soln adding about 5 μ L covers micelle, to prevent solution dry in enzymolysis process.Be placed in 37 DEG C of temperature-controlled box inside holding 12 ~ 16h; 5. extract: 0.1%TFA and 50% acetonitrile of the 60 μ L of the micelle after enzymolysis extract 3 times, each 20min, merge extract; 6. concentrate: drain under Vacuum Concentration instrument condition.
Adopt pepsin and elastoser to the heavy chain of monoclonal antibody IgG and light chain carry out in-gel digestion obtain polypeptide fragment time, itself and when adopting pancreatin to carry out in-gel digestion difference be: the damping fluid adopted when 4. enzymolysis is different, and that pepsin adopts when 4. enzymolysis is 5%TFA; When adopting elastoser to carry out 4. enzymolysis, damping fluid used is the Trisbuffer of pH=9.When pancreatin, pepsin and elastoser in-gel digestion except damping fluid during 4. enzymolysis is different, remaining parameter and operation steps are all the same.
4, enzyme cuts LC-MS/MS instrument on the polypeptide that obtains
Adopt after various enzyme completes in-gel digestion process, enzyme is cut the polypeptide fragment obtained and go up LC-MS/MS instrument respectively, described mass spectrometer is the mass spectrometer of various model, the TripleTOF5600-plus mass spectrometer of preferred ABSCIEX company, entering mass spectrometric enzyme, to cut polypeptide sample size be 5-15 μ L, is preferably 10 μ L.
5, NanoLC fingerprint figure analysis
NanoLC fingerprint figure analysis is that polypeptide sample enters mass spectrometer and needs after obtaining mass spectrometric data to analyze the heavy chain of IgG and the fingerprint image of light chain, the retention time of conserved sequence in IgG, electric charge, peak intensity and applied sample amount are carried out one_to_one corresponding, then determines the fingerprint image of monoclonal antibody IgG.The analysis of fingerprint image can lock the hypotype of IgG1/2/3.IgG has four kinds of hypotypes at least, the IgG1/2/3/4 of the such as mankind, the IgG1/2a/2b/3 etc. of mouse.The difference of different subtype is-COOH terminal sequence.Therefore, the singularity of monoclonal antibody order-checking is that IgG only only has the sequence in CDR1/2/3 region to be variable, and the sequence in other region is all conservative.Therefore, to compare common protein sequencing difficulty much lower for IgG protein sequencing.IgG comprises heavy chain (VDJ restructuring) and light chain (VJ restructuring) two long-chain polypeptide, and two polypeptide are connected together by disulfide bond and produce complete IgG antibody.After IgG enzymolysis, produce a lot of polypeptide, because conserved region is the same, therefore, it is consistent (at least closely similar) that the conserved region enzyme of same species cuts result.Different peptide sequences is from the polypeptide in CDR1/2/3 region, and these polypeptide show skew on the collection of illustrative plates of NanoLC, and the polypeptide of these skews is exactly CDR1/2/3 polypeptide.This is the key message of polypeptide NanoLC fingerprint image, the retention time of potential CDR1/2/3 sequence, peak intensity and m/z information (retention time data are in table one and table two) can be got by NanoLC fingerprint image, then can select these polypeptide specially and carry out MS/MS qualification.
Table one NGAL monoclonal antibody IgG heavy chain each enzymolysis spectrogram retention time
Table two NGAL monoclonal antibody IgG light chain each enzymolysis spectrogram retention time
6, spectrogram qualification and CDR1/2/3 qualification
When spectrogram qualification and CDR1/2/3 qualification, when the order-checking of monoclonal antibody IgG is reduced to CDR1/2/3 sequencing, monoclonal antibody IgG order-checking difficulty declines rapidly.Polypeptide spectrogram potential for CDR1/2/3 can be locked onto some region by the analysis of NanoLC fingerprint image, then these spectrograms of labor and obtain final CDR1/2/3 peptide sequence information.Then the conserved sequence in monoclonal antibody IgG and final CDR1/2/3 peptide sequence are carried out splicing and can obtain complete monoclonal antibody IgG amino acid sequence.Various enzyme cuts the heavy chain of NGAL monoclonal antibody and light chain spectrogram qualification result and polypeptide splicing result as shown in following table three and table four:
Table three NGAL monoclonal antibody IgG heavy chain each enzymolysis spectrogram qualification result
Table four NGAL monoclonal antibody IgG light chain each enzymolysis spectrogram qualification result
Use proteincoveragesummarizer software carries out polypeptide (peptide) and IgG antibody sequence carries out coverage rate analysis.Found that sequence of heavy chain almost successful matching completely (except Fc end has an amino acid to fail to match, but Fc is known array, has no effect to order-checking), namely variable region is surveyed logical completely; And sequence of light chain coverage rate is 97.66%, what fail that the match is successful is also the sequence of conserved region, and this sequence is also known array, and variable region is also complete tested logical.
It should be noted last that, above embodiment is only in order to illustrate implementer's case of this material and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
SEQUENCELISTING
<110> Shen He clouds
<120> monoclonal antibody IgG mass spectrum sequence measurement
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AlaCysGluValThrHisGlnGlyLeuSerSerProValThrLysSer
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Claims (5)
1. a monoclonal antibody IgG mass spectrum sequence measurement, is characterized in that, comprises the following steps:
(1) by monoclonal antibody IgG reductive alkylation;
(2) by electrophoresis method, the described monoclonal antibody IgG of reductive alkylation is separated heavy chain and light chain;
(3) heavy chain of in-gel digestion monoclonal antibody IgG and light chain obtain polypeptide fragment;
(4) enzyme is cut the polypeptide fragment obtained and is loaded onto LC-MS/MS instrument;
(5) CDR1/2/3 sequence is found out by NanoLC fingerprint map analyzing;
(6) CDR1/2/3 sequence.
2. monoclonal antibody IgG mass spectrum sequence measurement as claimed in claim 1, is characterized in that, the reductive alkylation in described step (1) adopts DTT-IAM pattern.
3. monoclonal antibody IgG mass spectrum sequence measurement as claimed in claim 1, it is characterized in that, the electrophoresis method in described step (2) is conventional SDS-PAGE electrophoresis.
4. monoclonal antibody IgG mass spectrum sequence measurement as claimed in claim 1, it is characterized in that, the heavy chain of the in-gel digestion monoclonal antibody IgG in described step (3) and light chain adopt conventional in-geldigestion method.
5. monoclonal antibody IgG mass spectrum sequence measurement as claimed in claim 1, is characterized in that, adding mass spectrometric enzyme in described step (4), to cut polypeptide sample size be 5-15 μ L.
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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 |
| CN107525934A (en) * | 2016-06-21 | 2017-12-29 | 张效龙 | A kind of method for determining antibodies in blood amino acid sequence |
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| CN107525934A (en) * | 2016-06-21 | 2017-12-29 | 张效龙 | A kind of method for determining antibodies in blood amino acid sequence |
| 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 |
| CN106645456B (en) * | 2016-10-13 | 2019-11-08 | 上海市食品药品检验所 | The sequencing approach of monoclonal antibody drug amino acid sequence is realized based on mass-spectrometric technique |
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