CN109932445A - A kind of evaluation method of a variety of charge isomer N sugar chain structures of glycoprotein - Google Patents
A kind of evaluation method of a variety of charge isomer N sugar chain structures of glycoprotein Download PDFInfo
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Abstract
The invention discloses a kind of evaluation methods of a variety of charge isomer N sugar chain structures of glycoprotein.Method includes the following steps: the separation of charge heteroplasmon, the collection of charge heteroplasmon, in-gel digestion, sugar chain enrichment, sugar chain label, sugar chain purifying, mass spectral analysis.Present invention discover that isoelectric focusing electrophoresis (IEF) separates the means of a variety of charge isomers and in-gel digestion for analyzing N sugar chain structure, a variety of charge isomers are overcome to the difficult point of N sugar chain structural analysis, it is capable of the N sugar chain structure of the single charge glycoprotein of Accurate Analysis, it is more advantageous to and Accurate Analysis is carried out to a variety of charge isomer N sugar structures, the analysis and evaluation suitable for a variety of charge heteroplasmon N sugar chain structures of glycoprotein.
Description
Technical field
The present invention relates to protein medical bioengineering and technical fields, are related to a kind of a variety of charge isomer N of glycoprotein
The evaluation method of sugar chain structure.More particularly to a kind of recombined human vascular endothelial growth factor receptor Fc(VEGFR-Fc) fusion
The evaluation method of a variety of charge isomer N sugar chain structures of albumen.
Background technique
Glycosylation is modified after a kind of most common protein translation, according to the difference of connection type, can be divided into N- glycosylation
It is glycosylated with O-, wherein N glycosylation is sugar chain and coding protein amino acid sequence Asn-X-Ser/Thr(X is in addition to Pro
Amino acid) in-NH on Asn residue2It is connected, O- glycosylation is on sugar chain and coding protein amino acid sequence Ser/Thr residue
- OH be connected.Currently, human cytokines drug is mostly expressed by gene-recombinated cell, type of glycosylation and level are to drug
Drug effect, half-life period, stability and safety etc. in vivo has different degrees of influence.Such as monoclonal antibody or fusion
Albumen, the lower Sialic Acid Level of high mannose sugar-type and end, can all influence pharmacodynamics (pK), cause intracorporal fast
Speed is removed, half life；Core fucose can reduce the affinity with receptor, lead to the thin of antibody dependent cellular mediation
Cellular toxicity (ADCC) activity decline；Terminal galactose mainly influences cytotoxicity (CDC) activity of Complement Dependent.
Recombinant human VEGF R-Fc fusion protein is homodimer glycoprotein, by Chinese hamster ovary cell (Chinese hamster ovary celI)
Expression, and obtained through highly purified and viral inaction steps.The albumen contains multiple glycosylation modified sites, has a variety of charges
Isomers, sugar-type is extremely complex, for the consistency for guaranteeing each production batch sugar-type, needs stable production technology and effective sugar
Type analysis method.In order to further carry out quality research to product, and purification process is finely controlled, improves product matter
Amount is separated herein by a variety of charge heteroplasmons of Isoelectric Focusing swimmer's glycoprotein, and to isolated isomers digestion, extract and
Sugar chain is marked, fluorescence mass spectral analysis is carried out in conjunction with sugar chain of the LC-MS technology to label, for instructing purpose in purification process
The collection of albumen achievees the purpose that obtain high quality glycoprotein.
Currently, sugar chain structural analysis carries out digestion processing to albumen after mainly using glycoprotein denaturation reduction in glycoprotein,
It is then enriched with sugar chain and carries out fluorescent marker, label sugar chain is further purified, hydrophilic chromatographic column is used in combination to various sugar chains point
From progress fluorescence mass spectral analysis.And this method uses the very high a variety of charge isomers of Isoelectric Focusing swimmer of resolution ratio first
Then isolated sugar chain is cut respectively using the method for in-gel digestion, then carries out sugar chain enrichment and fluorescent marker by separation, into one
Purification tag sugar chain is walked, hydrophilic chromatographic column is used in combination, various sugar chains is separated, finally carry out fluorescence mass spectral analysis.This has
Help carry out sugar chain structural analysis respectively to a variety of charge isomers, it is poor to facilitate the sugar-type studied between different charge isomers
It is different, it is more advantageous in production process and quality control is carried out to different charge isomers.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of evaluations of a variety of charge isomer N sugar chain structures of glycoprotein
Method, the method overcome a variety of charge isomers to the difficult point of N sugar chain structural analysis, can be to a variety of charge isomeries of glycoprotein
Body N sugar chain structure is analyzed, and is capable of the N sugar chain structure of the single charge glycoprotein of Accurate Analysis, is facilitated mesh in purification process
Albumen collection, achieve the purpose that obtain high quality glycoprotein.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
A kind of evaluation method of a variety of charge isomer N sugar chain structures of glycoprotein.The following steps are included: (1) passes through isoelectric focusing
Electrophoresis separates a variety of charge heteroplasmons, and the gel after electrophoresis is fixed and is dyed；(2) to a variety of electricity after separation
Lotus heteroplasmon band number, cuts and is collected into centrifuge tube；(3) multiple gel-tape samples after collection are decolourized, then into
Row reduction and alkylation processing, finally carry out in-gel digestion using glycosidase F；(4) after digestion, after extracting in-gel digestion
Sugar chain precipitates the albumen in solution using organic solvent, collects supernatant and drying, is enriched with sugar chain；(5) formic acid is used
It handles the sugar chain of enrichment and is dried, fluorescence mark then is carried out using 2- aminobenzamide (2-AB) to dry sugar chain
Note；(6) sugar chain of label is purified using N sugar purifying pillar, collects label sugar chain；(7) sugar chain after label is carried out
Fluorescence mass spectral analysis confirms N sugar chain structure.
Wherein the glycoprotein is the glycoprotein with multiple glycosylation sites.
Wherein the glycoprotein has a variety of charge isomers.
Wherein the glycoprotein is recombination human VEGFR-3-Fc fusion protein.
Wherein isoelectric focusing electrophoresis (IEF) is using horizontally or vertically isoelectric focusing electrophoresis in the step 1, wherein it is preferred that hanging down
Straight isoelectric focusing electrophoresis.
Wherein gel used in isoelectric focusing electrophoresis is IEF gel in step 1, and fixer is solution of trichloroacetic acid, dyeing liquor
For coomassie brilliant blue staining liquid.
In a specific embodiment, isoelectric focusing electrophoresis may be accomplished by: take appropriate recombinant human VEGF R-
Fc fusion protein changes liquid into a certain amount of ultrapure water with super filter tube ultrafiltration；Take sample and 2 × sample buffer etc. after appropriate ultrafiltration
Loading after volume mixes, gel used are pH3-10 IEF gel；Deposition condition is 100V electrophoresis 60min, 200V electrophoresis
60min, 500V electrophoresis 30min.
Reduction treatment is carried out to glycoprotein using dithiothreitol (DTT) (DTT) solution in the step of wherein described 3；DTT is added
The concentration of solution is 0.5M-1M, and the final concentration of 2-25mM of DTT of reduction treatment is carried out to albumen；The end of further preferred DTT is dense
Spend 5-20mM；DTT final concentration of 2 mM, 10mM or 25mM in some embodiments.
Wherein, be alkylated processing using iodoacetamide (IAM) in step 3, used in the concentration of IAM be 5-
62.5 mM, the concentration of IAM is 5Mm, 25 mM, 62.5mM in some embodiments.
Glycosidase F(PNGase F used in the step of wherein described 3) final concentration of 2-20U/ μ l；Further preferably
The final concentration of 4-10U/ μ l of PNGase F；Final concentration of 2U/ μ l, the 5U/ μ l or 20 of PNGase F in some embodiments
U/μl。
Extraction solution used is 50% acetonitrile solution containing formic acid, acetic acid or trifluoroacetic acid in the step of wherein described 4；It is excellent
Select 50% acetonitrile solution containing formic acid or trifluoroacetic acid；More preferably 50% acetonitrile solution containing formic acid.
50% acetonitrile solution that solution is the formic acid containing 0.1%-5% is extracted in certain embodiments；Further preferably contain 0.5%-
50% acetonitrile solution of 2% formic acid；More preferably contain 50% acetonitrile solution of 1% formic acid.
Organic solvent used in albumen precipitation has methanol, ethyl alcohol or butanol, preferred alcohol or fourth in the step of wherein described 4
Alcohol, more preferable ethyl alcohol.
Wherein the volume ratio of organic solvent used is 50%-90%, and further preferred 60%-80% has in certain embodiments
The volume ratio of solvent is 50%, 75% or 90%.
The volume ratio of ethyl alcohol used is 75% in certain embodiments.
Wherein sugar chain is purified using GlycoWorks HILIC 1cc pillar in step 6.In a specific embodiment party
In formula, LC-MS uses Waters H-Class G2-XS QTof system, then progress QTof mass spectral analysis, data processing,
Wherein chromatographic column use Waters ACQUITY UPLC Glycan BEH Amide(130,1.7 μm, 2.1 × 150mm).
The present invention is separated by using a variety of charge isomers of the very high Isoelectric Focusing swimmer of resolution ratio, and to gel strips
Band is collected, and reduces the influence of a variety of charge isomers in the analysis of N sugar chain, is conducive to the N sugar to single charge glycoprotein
Chain structure is analyzed and evaluated.
The present invention cuts sugar chain by using the method for in-gel digestion, reduces in glue and carries out again after recycling, purifying protein
The tedious steps such as digestion are not required to expensive consumptive material and equipment, easy to operate, can it is simple, quickly finish digestion and collect sugar
Chain.
The present invention is enriched with by sugar chain and fluorescent marker, and label sugar chain is further purified, and hydrophilic chromatographic column pair is used in combination
Various sugar chains are separated, then carry out fluorescence mass spectral analysis.The present invention compared with the conventional method, firstly, present invention employs point
It is other that sugar chain structural analysis is carried out to a variety of charge isomers, help to study the sugar-type difference between different charge isomers, more
Be conducive to carry out quality control to different charge isomers in production process, to the glycoprotein with a variety of charge isomers
N sugar chain structural analysis has good reference, secondly, present invention employs carry out sugar chain to a variety of charge isomers respectively
Structural analysis facilitates the glycoprotein for instructing selective collection to have hypotoxicity, high curative effect in process of production, effectively improves sugar
The clinical application of albumen.
Detailed description of the invention
Fig. 1: the IEF electrophorogram of recombinant human VEGF R-Fc fusion protein analysis sample.
Specific embodiment
In order to which the present invention is further explained, below in conjunction with specific embodiments of the present invention, in the embodiment of the present invention
Technical method is clearly and completely described.Unless otherwise specified, reagent involved in the embodiment of the present invention is commercially available production
Product can be bought by commercial channel and be obtained.
Embodiment 1, recombinant human VEGF R-Fc fusion protein charge isomer N sugar chain structural analysis
(1) IEF separates charge heteroplasmon
Recombinant human VEGF R-Fc fusion protein 1mg is taken, with 10kD, 0.5ml super filter tube ultrafiltration changes liquid into 100 μ l ultrapure waters, enzyme
It is quantitative to mark instrument protein concentration.Loading after sample and 2 × sample buffer mix in equal volume after appropriate ultrafiltration is taken, albumen applied sample amount is
100 μ g/ bands, gel used are pH3-10 IEF gel.Deposition condition is as follows.
The setting of 1 IEF electrophoretic voltage of table
Electrophoresis finishes, and by gel with ultrapure water 2 times, is then immersed in 12% trichloroacetic acid fixer and is fixed, room temperature
Shake 30min.After gel is cleaned 3 times with ultrapure water later, appropriate coomassie brilliant blue staining liquid is added, shakes at room temperature
30min is obvious, clear to band.Gel is put into ultrapure water again and is shaken, 10min replace a ultrapure water to gel background without
Obvious color, electrophorogram is as shown in Figure 1.
(2) in-gel digestion
1. charge isomer is collected
Gel-tape after electrophoresis is labeled as 1-12 from top to bottom (pI from high to low), is cut each band respectively using scalpel
Under, and it is cut into 1 × 1mm fritter, it is respectively charged into 1.5ml centrifuge tube；The de- of the 25mM ammonium hydrogen carbonate containing 50% acetonitrile is added in every pipe
300 μ l of color solution, room temperature concussion 30min decolourize, and decoloration 3-4 times are repeated, until gel color takes off to colourless or very light；It inhales
300 μ l of acetonitrile is added in destainer out, places 10min, and acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge.
2. reduction and alkylation
The 300 μ l of 50mM ammonium bicarbonate soln of the DTT containing 10mM is added in every pipe, in 56 DEG C of water-bath 30min, DTT solution is sucked out, adds
Enter 300 μ l of acetonitrile, place 10min, acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge；Then every pipe addition contains
The 300 μ l of 50mM ammonium bicarbonate soln of 25mM IAM, room temperature are protected from light 45min, and IAM solution is sucked out, and 300 μ l of acetonitrile is added,
10min is placed, acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge.
3. in-gel digestion
The 300 μ l of 50mM ammonium bicarbonate soln of the l of μ containing 5U/ PNGase F glycosidase is added in every pipe, in 37 DEG C of water-bath digestion 15h.
(3) extract and be enriched with sugar chain
1. extracting sugar chain
Digestion finishes, Aspirate supernatant, collects into new 1.5ml centrifuge tube；It is added in remaining micelle and contains the 50% of 1% formic acid
30min is extracted in 300 μ l of acetonitrile solution, room temperature concussion, and Aspirate supernatant merges with collection liquid before, repeats to extract 2 times, and will most
The collection liquid merged eventually is concentrated into about 300 μ l in freeze concentration centrifuge.
2. being enriched with sugar chain
By above-mentioned sample, the 900 μ l of ice ethyl alcohol of -30 DEG C of preservations are added, mix, in -30 DEG C of alcohol precipitation 15min, then with low temperature from
Scheming, under the conditions of 4 DEG C, 13000rpm is centrifuged 15min；Supernatant after taking centrifugation, has been concentrated into freeze concentration centrifuge
White drying.
(4) sugar chain is marked
1. marking pre-treatment
50% acetonitrile solution, the 100 μ l for containing 1% formic acid is added in every pipe, mixes, and is incubated at room temperature 40min, is then centrifuged in freeze concentration
Dry sugar chain in machine.
2. 2-AB marks sugar chain
2-AB label reaction solution is added in the dry sugar chain of every pipe (to weigh 2-AB 100mg, the DMSO solution for containing 30% acetic acid is added
2ml mixes, adds sodium cyanoborohydride 120mg, mixes, i.e. 2-AB marks reaction solution) 60 μ l, it mixes, 65 DEG C of reaction 3h.
(5) sugar chain is purified
Using the small column purification sugar chain of GlycoWorks HILIC 1cc, specific steps are as follows:
1. 1ml ultrapure water is added in each pillar to be balanced, ultrapure water is extracted out with vacuum pump, adds 85% acetonitrile of 1ml
Solution is extracted out 85% acetonitrile solution with vacuum pump.
2. 600 μ l acetonitriles is taken to be added in the sugar chain sample of 60 μ l 2-AB label, and loading is carried out, will be marked with vacuum pump
Remember reagent extraction, then wash column with 500 μ l, 85% acetonitrile solution, is extracted out above-mentioned solution with vacuum pump.
3. 25mM ammonium bicarbonate soln elution sugar chain of the 100 μ l containing 5% acetonitrile is added, eluent is extracted out simultaneously with vacuum pump
It collects, repeats elution 4 times, eluent is concentrated to dryness in freeze concentration centrifuge.
(6) UPLC-ESI-Q-TOF is identified
Each sample is redissolved in appropriate ultrapure water, is mixed, before mass spectral analysis, appropriate acetonitrile (second into the sugar chain redissolved is added
Nitrile is final concentration of 60%).Through Waters ACQUITY UPLC Glycan BEH Amide(130,1.7 μm, 2.1 × 150mm)
After separation, ESI-Q-TOF Mass Spectrometer Method, the analysis of Unifi software.The UPLC-ESI-Q-TOF analysis parameter includes liquid phase ginseng
Number, mass spectrum acquisition parameter and sample analysis parameter are shown in Table 2 ~ 4.
2 liquid phase parameter of table
3 mass spectrum acquisition parameter of table
4 sample analysis parameter of table
(7) experimental result
The Dextran Ladder standard items (homopolymer of glucose of 2-AB label) marked using 2-AB are selected as calibration standard product
Taking the degree of polymerization is the chromatographic peak of 4 to 15 glucose units, and it is bent that retention time and GU value are fitted to five power polynomial calibrations
Line.According to the essence in the retention time (being indicated with glucose unit, also referred to as GU) and Waters oligosaccharides GU database after correction
True mass number carries out automatic sugar-type matching to the N sugar chain of 2-AB label, responds further according to N sugar chain each in sample and total N sugar chain
Value calculates each N sugar chain proportion in total N sugar chain, and the results are shown in Table 5.
Different sugar chain structure proportion results in each band of 5 IEF of table (pI from high to low)
Nomenclature explanation: A represents a N-Acetyl-D-glucosamine and a galactose units on pentasaccharides core space, and G is represented
Galactolipin, S represent sialic acid, and F represents salt algae sugar, and M represents mannose.
Embodiment 2, high pI recombinant human VEGF R-Fc fusion protein charge isomer N sugar chain structural analysis
(1) IEF separates charge heteroplasmon
High pI recombinant human VEGF R-Fc fusion protein 1mg is taken, with 10kD, 0.5ml super filter tube ultrafiltration changes liquid to 100 μ l ultrapure waters
In, microplate reader protein concentration is quantitative.Loading after sample and 2 × sample buffer mix in equal volume after appropriate ultrafiltration is taken, on albumen
Sample amount is 100 μ g/ bands, and gel used is pH3-10 IEF gel.Deposition condition is as follows.
The setting of 1 IEF electrophoretic voltage of table
Electrophoresis finishes, and by gel with ultrapure water 2 times, is then immersed in 12% trichloroacetic acid fixer and is fixed, room temperature
Shake 30min.After gel is cleaned 3 times with ultrapure water later, appropriate coomassie brilliant blue staining liquid is added, shakes at room temperature
30min is obvious, clear to band.Gel is put into ultrapure water again and is shaken, 10min replace a ultrapure water to gel background without
Obvious color, electrophorogram is as shown in Figure 1.
(2) in-gel digestion
1. charge isomer is collected
Gel-tape after electrophoresis is labeled as 1-12 from top to bottom (pI from high to low), is cut each band respectively using scalpel
Under, and it is cut into 1 × 1mm fritter, it is respectively charged into 1.5ml centrifuge tube；The de- of the 25mM ammonium hydrogen carbonate containing 50% acetonitrile is added in every pipe
300 μ l of color solution, room temperature concussion 30min decolourize, and decoloration 3-4 times are repeated, until gel color takes off to colourless or very light；It inhales
300 μ l of acetonitrile is added in destainer out, places 10min, and acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge.
2. reduction and alkylation
The 300 μ l of 50mM ammonium bicarbonate soln of the DTT containing 2mM is added in every pipe, and in 56 DEG C of water-bath 30min, DTT solution is sucked out, is added
300 μ l of acetonitrile places 10min, and acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge；Then every pipe, which is added, contains 5mM
The 300 μ l of 50mM ammonium bicarbonate soln of IAM, room temperature are protected from light 45min, and IAM solution is sucked out, and 300 μ l of acetonitrile is added, and place
Acetonitrile is sucked out in 10min, and the dry blob of viscose in freeze concentration centrifuge.
3. in-gel digestion
The 300 μ l of 50mM ammonium bicarbonate soln of the l of μ containing 2U/ PNGase F glycosidase is added in every pipe, in 37 DEG C of water-bath digestion 15h.
(3) extract and be enriched with sugar chain
1. extracting sugar chain
Digestion finishes, Aspirate supernatant, collects into new 1.5ml centrifuge tube；It is added in remaining micelle containing 0.5% formic acid
30min is extracted in 50% acetonitrile solution, 300 μ l, room temperature concussion, and Aspirate supernatant merges with collection liquid before, repeats to extract 2 times, and
The collection liquid finally merged is concentrated into about 300 μ l in freeze concentration centrifuge.
2. being enriched with sugar chain
By above-mentioned sample, the 900 μ l of ice methanol of -30 DEG C of preservations are added, mix, in -30 DEG C of alcohol precipitation 15min, then with low temperature from
Scheming, under the conditions of 4 DEG C, 13000rpm is centrifuged 15min；Supernatant after taking centrifugation, has been concentrated into freeze concentration centrifuge
White drying.
(4) sugar chain is marked
1. marking pre-treatment
50% acetonitrile solution, the 100 μ l for containing 1% formic acid is added in every pipe, mixes, and is incubated at room temperature 40min, is then centrifuged in freeze concentration
Dry sugar chain in machine.
2. 2-AB marks sugar chain
2-AB label reaction solution is added in the dry sugar chain of every pipe (to weigh 2-AB 100mg, the DMSO solution for containing 30% acetic acid is added
2ml mixes, adds sodium cyanoborohydride 120mg, mixes, i.e. 2-AB marks reaction solution) 60 μ l, it mixes, 65 DEG C of reaction 3h.
(5) sugar chain is purified
Using the small column purification sugar chain of GlycoWorks HILIC 1cc, specific steps are as follows:
1. 1ml ultrapure water is added in each pillar to be balanced, ultrapure water is extracted out with vacuum pump, adds 85% acetonitrile of 1ml
Solution is extracted out 85% acetonitrile solution with vacuum pump.
2. 600 μ l acetonitriles is taken to be added in the sugar chain sample of 60 μ l 2-AB label, and loading is carried out, will be marked with vacuum pump
Remember reagent extraction, then wash column with 500 μ l, 85% acetonitrile solution, is extracted out above-mentioned solution with vacuum pump.
3. 25mM ammonium bicarbonate soln elution sugar chain of the 100 μ l containing 5% acetonitrile is added, eluent is extracted out simultaneously with vacuum pump
It collects, repeats elution 4 times, eluent is concentrated to dryness in freeze concentration centrifuge.
(6) UPLC-ESI-Q-TOF is identified
Each sample is redissolved in appropriate ultrapure water, is mixed, before mass spectral analysis, appropriate acetonitrile (second into the sugar chain redissolved is added
Nitrile is final concentration of 60%).Through Waters ACQUITY UPLC Glycan BEH Amide(130,1.7 μm, 2.1 × 150mm)
After separation, ESI-Q-TOF Mass Spectrometer Method, the analysis of Unifi software.The UPLC-ESI-Q-TOF analysis parameter includes liquid phase ginseng
Number, mass spectrum acquisition parameter and sample analysis parameter are the same as " embodiment 1, recombinant human VEGF R-Fc fusion protein charge isomer N sugar chain
Structural analysis " table 2 ~ 4.
(7) experimental result
The Dextran Ladder standard items (homopolymer of glucose of 2-AB label) marked using 2-AB are selected as calibration standard product
Taking the degree of polymerization is the chromatographic peak of 4 to 15 glucose units, and it is bent that retention time and GU value are fitted to five power polynomial calibrations
Line.According to the essence in the retention time (being indicated with glucose unit, also referred to as GU) and Waters oligosaccharides GU database after correction
True mass number carries out automatic sugar-type matching to the N sugar chain of 2-AB label, responds further according to N sugar chain each in sample and total N sugar chain
Value calculates each N sugar chain proportion in total N sugar chain, and the results are shown in Table 6.
Different sugar chain structure proportion results in each band of 6 IEF of table (pI from high to low)
Embodiment 3, low pI recombinant human VEGF R-Fc fusion protein charge isomer N sugar chain structural analysis
(1) IEF separates charge heteroplasmon
Low pI recombinant human VEGF R-Fc fusion protein 1mg is taken, with 10kD, 0.5ml super filter tube ultrafiltration changes liquid to 100 μ l ultrapure waters
In, microplate reader protein concentration is quantitative.Loading after sample and 2 × sample buffer mix in equal volume after appropriate ultrafiltration is taken, on albumen
Sample amount is 100 μ g/ bands, and gel used is pH3-10 IEF gel.Deposition condition is as follows.
The setting of 1 IEF electrophoretic voltage of table
Electrophoresis finishes, and by gel with ultrapure water 2 times, is then immersed in 12% trichloroacetic acid fixer and is fixed, room temperature
Shake 30min.After gel is cleaned 3 times with ultrapure water later, appropriate coomassie brilliant blue staining liquid is added, shakes at room temperature
30min is obvious, clear to band.Gel is put into ultrapure water again and is shaken, 10min replace a ultrapure water to gel background without
Obvious color, electrophorogram is as shown in Figure 1.
(2) in-gel digestion
1. charge isomer is collected
Gel-tape after electrophoresis is labeled as 1-10 from top to bottom (pI from high to low), is cut each band respectively using scalpel
Under, and it is cut into 1 × 1mm fritter, it is respectively charged into 1.5ml centrifuge tube；The de- of the 25mM ammonium hydrogen carbonate containing 50% acetonitrile is added in every pipe
300 μ l of color solution, room temperature concussion 30min decolourize, and decoloration 3-4 times are repeated, until gel color takes off to colourless or very light；It inhales
300 μ l of acetonitrile is added in destainer out, places 10min, and acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge.
2. reduction and alkylation
The 300 μ l of 50mM ammonium bicarbonate soln of the DTT containing 25mM is added in every pipe, in 56 DEG C of water-bath 30min, DTT solution is sucked out, adds
Enter 300 μ l of acetonitrile, place 10min, acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge；Then every pipe addition contains
The 300 μ l of 50mM ammonium bicarbonate soln of 62.5mM IAM, room temperature are protected from light 45min, and IAM solution is sucked out, and 300 μ of acetonitrile is added
L places 10min, and acetonitrile is sucked out, and the dry blob of viscose in freeze concentration centrifuge.
3. in-gel digestion
The 300 μ l of 50mM ammonium bicarbonate soln of the l of μ containing 20U/ PNGase F glycosidase is added in every pipe, in 37 DEG C of water-bath digestion 15h.
(3) extract and be enriched with sugar chain
1. extracting sugar chain
Digestion finishes, Aspirate supernatant, collects into new 1.5ml centrifuge tube；It is added in remaining micelle and contains the 50% of 2% formic acid
30min is extracted in 300 μ l of acetonitrile solution, room temperature concussion, and Aspirate supernatant merges with collection liquid before, repeats to extract 2 times, and will most
The collection liquid merged eventually is concentrated into about 300 μ l in freeze concentration centrifuge.
2. being enriched with sugar chain
By above-mentioned sample, the 900 μ l of ice butanol of -30 DEG C of preservations are added, mix, in -30 DEG C of alcohol precipitation 15min, then with low temperature from
Scheming, under the conditions of 4 DEG C, 13000rpm is centrifuged 15min；Supernatant after taking centrifugation, has been concentrated into freeze concentration centrifuge
White drying.
(4) sugar chain is marked
1. marking pre-treatment
50% acetonitrile solution, the 100 μ l for containing 1% formic acid is added in every pipe, mixes, and is incubated at room temperature 40min, is then centrifuged in freeze concentration
Dry sugar chain in machine.
2. 2-AB marks sugar chain
2-AB label reaction solution is added in the dry sugar chain of every pipe (to weigh 2-AB 100mg, the DMSO solution for containing 30% acetic acid is added
2ml mixes, adds sodium cyanoborohydride 120mg, mixes, i.e. 2-AB marks reaction solution) 60 μ l, it mixes, 65 DEG C of reaction 3h.
(5) sugar chain is purified
Using the small column purification sugar chain of GlycoWorks HILIC 1cc, specific steps are as follows:
1. 1ml ultrapure water is added in each pillar to be balanced, ultrapure water is extracted out with vacuum pump, adds 85% acetonitrile of 1ml
Solution is extracted out 85% acetonitrile solution with vacuum pump.
2. 600 μ l acetonitriles is taken to be added in the sugar chain sample of 60 μ l 2-AB label, and loading is carried out, will be marked with vacuum pump
Remember reagent extraction, then wash column with 500 μ l, 85% acetonitrile solution, is extracted out above-mentioned solution with vacuum pump.
3. 25mM ammonium bicarbonate soln elution sugar chain of the 100 μ l containing 5% acetonitrile is added, eluent is extracted out simultaneously with vacuum pump
It collects, repeats elution 4 times, eluent is concentrated to dryness in freeze concentration centrifuge.
(6) UPLC-ESI-Q-TOF is identified
Each sample is redissolved in appropriate ultrapure water, is mixed, before mass spectral analysis, appropriate acetonitrile (second into the sugar chain redissolved is added
Nitrile is final concentration of 60%).Through Waters ACQUITY UPLC Glycan BEH Amide(130,1.7 μm, 2.1 × 150mm)
After separation, ESI-Q-TOF Mass Spectrometer Method, the analysis of Unifi software.The UPLC-ESI-Q-TOF analysis parameter includes liquid phase ginseng
Number, mass spectrum acquisition parameter and sample analysis parameter are the same as " embodiment 1, recombinant human VEGF R-Fc fusion protein charge isomer N sugar chain
Structural analysis " table 2 ~ 4.
(7) experimental result
The Dextran Ladder standard items (homopolymer of glucose of 2-AB label) marked using 2-AB are selected as calibration standard product
Taking the degree of polymerization is the chromatographic peak of 4 to 15 glucose units, and it is bent that retention time and GU value are fitted to five power polynomial calibrations
Line.According to the essence in the retention time (being indicated with glucose unit, also referred to as GU) and Waters oligosaccharides GU database after correction
True mass number carries out automatic sugar-type matching to the N sugar chain of 2-AB label, responds further according to N sugar chain each in sample and total N sugar chain
Value calculates each N sugar chain proportion in total N sugar chain, and the results are shown in Table 7.
Different sugar chain structure proportion results in each band of 7 IEF of table (pI from high to low)
Claims (11)
1. a kind of evaluation method of a variety of charge isomer N sugar chain structures of glycoprotein, method includes the following steps:
(1) charge heteroplasmon separates: being separated by a variety of charge heteroplasmons of Isoelectric Focusing swimmer, to the gel after electrophoresis
It is fixed and dyes；
(2) charge heteroplasmon is collected: being numbered to a variety of charge heteroplasmon bands after separation, is cut and be collected into centrifuge tube；
(3) in-gel digestion: multiple gel-tape samples after collection are decolourized, and restore again and alkylation is handled, finally make
In-gel digestion is carried out with glycosidase F；
(4) sugar chain be enriched with: after digestion, extract in-gel digestion after sugar chain, using organic solvent to the albumen in solution into
Row precipitating, collects supernatant and drying, is enriched with sugar chain；
(5) sugar chain marks: handling the sugar chain of enrichment using formic acid and is dried, then uses 2- aminobenzene to dry sugar chain
Formamide (2-AB) carries out fluorescent marker；
(6) sugar chain purifies: being purified using N sugar purifying pillar to the sugar chain of label, collects label sugar chain；
(7) mass spectral analysis: fluorescence mass spectral analysis is carried out to the sugar chain after label, confirms N sugar chain structure.
2. according to the method described in claim 1, it is characterized in that wherein glycoprotein be recombination human vascular endothelial growth because
Sub- Receptor Fc Fusion Proteins.
3. according to the method described in claim 1, it is characterized in that wherein being coagulated used in isoelectric focusing electrophoresis as described in step (1)
Glue is IEF gel, and fixer is solution of trichloroacetic acid, and dyeing liquor is coomassie brilliant blue staining liquid.
4. according to the method described in claim 1, it is characterized in that wherein isoelectric focusing electrophoresis as described in step (1) uses water
Ordinary telegram focusing electrophoresis or vertical isoelectric focusing electrophoresis.
5. according to the method described in claim 1, it is characterized in that wherein in step (3) using dithiothreitol (DTT) to glycoprotein into
Row reduction treatment is alkylated processing to glycoprotein using iodoacetamide.
6. according to the method described in claim 4, it is characterized in that the concentration of dithiothreitol (DTT) solution is wherein added in step (3)
For 0.5M-1M, the final concentration of 2-25mM of DTT of reduction treatment is carried out to albumen, the concentration of iodoacetamide is 5-62.5 mM.
7. according to the method described in claim 1, it is characterized in that wherein using the final concentration of 2- of glycosidase F in step (3)
20U/μl。
8. according to the method described in claim 1, it is characterized in that molten using 50% acetonitrile containing formic acid, acetic acid or trifluoroacetic acid
Liquid extracts sugar chain, wherein preferably comprising 50% acetonitrile solution of formic acid.
9. according to the method described in claim 1, it is characterized in that the organic solvent in step (4) is methanol, ethyl alcohol or butanol,
Wherein preferred alcohol.
10. according to the method described in claim 1, it is characterized in that step (6) uses GlycoWorks HILIC 1cc pillar
Sugar chain is purified.
11. according to the method described in claim 1, it is characterized in that step (7) uses ultra high efficiency liquid phase to sugar chain after purification
Chromatographic tandem electron spray-quadrupole time-of-flight mass spec-trometry method carries out data acquisition, and mass spectrum software is combined to carry out data analysis.
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