CN107793496A - A kind of general glycoprotein N sugar chain method for releasing - Google Patents
A kind of general glycoprotein N sugar chain method for releasing Download PDFInfo
- Publication number
- CN107793496A CN107793496A CN201710984428.8A CN201710984428A CN107793496A CN 107793496 A CN107793496 A CN 107793496A CN 201710984428 A CN201710984428 A CN 201710984428A CN 107793496 A CN107793496 A CN 107793496A
- Authority
- CN
- China
- Prior art keywords
- glycoprotein
- sugar chains
- sugar chain
- sugar
- phase extraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0033—Xanthan, i.e. D-glucose, D-mannose and D-glucuronic acid units, saubstituted with acetate and pyruvate, with a main chain of (beta-1,4)-D-glucose units; Derivatives thereof
Abstract
The invention belongs to biological technical field, specifically disclosing a kind of general glycoprotein N sugar chains method for releasing includes glycoprotein sample being dissolved in concentrated ammonia liquor, 55 70 DEG C of 20h of water-bath 12 in closed container, obtained reaction solution is concentrated under reduced pressure drying, water is added to dissolve again, obtained N sugar chains crude product solution adjusts pH to neutrality, then in turn through the purifying of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column, completes the release and preparation of N sugar chains.This method is versatile, is had the advantage that compared with having method at present：Suitable for neutral N sugar chains, acid N sugar chains and the N sugar chains modified containing the fucoses of core α 1,3；The micro-analysis and can that sugar chain can be used for is used for the extensive preparation of sugar chain；Operating procedure is simple, quick, and cost is cheap, overcomes traditional enzyme process and the defects of the chemical method reported in the past discharges glycoprotein N sugar chains；The reproducibility N sugar that the method obtains can directly carry out analyzing and can be used for a variety of derivatization method derivatization post analysis.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of general glycoprotein N- sugar chain method for releasing.
Background technology
Glycosylation is a kind of one of universal protein post-translational modification, plays and focuses in the regulation and control of cell activities
Function is wanted, such as participates in the occurrence and development of cell adherence, signal transduction, Immune discrimination and disease.The property and work(of glycoprotein
Can be closely related with the structure of sugar chain.And glycoprotein structure is complicated, molecular weight is big, it is difficult to directly be analyzed and researched, typically
Need to be analyzed after sugar chain is discharged from albumen, therefore, the release of sugar chain is one in sugar chain analysis and closed very much
The link of key, the analysis and research to glycoprotein candy chain 26S Proteasome Structure and Function are significant.
At present, the method for releasing of N- sugar chains mainly has enzyme process and the major class of chemical method two.Enzymatic isolation method is mainly special using height
The glycosidase or proteolytic enzyme of the opposite sex carry out digestion, so as to obtaining free sugar chain.Enzyme used mainly has N- glycosidases F
(PNGaseF), N- glycosidases A (PNGasA), endoglycosidase H (EndoH) etc., these are all the strong specific glucosides of selectivity
Enzyme, they can identify specific glycosidic bond.Wherein, PNGaseF enzymolysis is a kind of the most frequently used method, and it can effectively discharge
Most of N- sugar chains, but the enzyme can not discharge plant, lower animal or the modification of microbe-derived core α -1,3- fucose
N- sugar chains.Although PNGase A enzymes can discharge α -1, the N- sugar chains of 3- fucoses modification, reaction efficiency is relatively low, uncomfortable
Large-scale use is closed, and this enzyme is expensive, is only applicable to micro-analysis, it is difficult to for large-scale sugar group credit analysis.Separately
Outside, the N- sugar chains of non-pentasaccharides core texture are also widely present in nature, as N- sugar chains lack on the basis of pentasaccharides core
One mannose or a more N-Acetyl-D-glucosamine, and some are connected on polypeptide by reducing end of high mannose
N- sugar chains etc., above two enzyme can not all discharge, therefore enzyme process has some limitations.
Common chemical method has hydrazinolysis method, i.e. glycoprotein can be with the release of irreducibility in 90 DEG C of reaction 4h in anhydrous hydrazine
Sugar chain, but anhydrous hydrazine has severe toxicity, and deacetylation occurs.Research finds glycoprotein in 1M NaOH-1M NaBH4In system
4-6h is reacted in 100 DEG C, reproducibility N- sugar chains can be dissociateed, but follow-up separation point can not be unfavorable for its fluorescent derivatization
Analysis.We have developed N- sugar chain irreducibility method for releasing, i.e. glycoprotein reacts 16h in NaOH solution in 50 DEG C, can discharge
Without core α -1, the N- sugar chains of 3- fucoses, without accessory substance, but the N- sugar chain meetings of core α -1,3- fucose modification
Generation peeling degrades.Also the method for having the sodium hypochlorite release N- sugar chains of research and development can be used for preparing on a large scale, but
Release efficiency is relatively low, and accessory substance is more in reaction, and miscellaneous peak background is higher during micro mass spectral analysis N- sugar chains.So lack at present logical
Chemical method discharges N- sugar chains.
To sum up, subject matter existing for prior art is, enzyme process prepares N- sugar chains, and selectivity to be present strong, costly, changes
Method lacks versatility.Therefore, the new side of glycosylation N- sugar chains release dissociation versatile, easy to operate, cost is cheap is developed
Method, to N- sugar chains analyze and identify and prepare it is significant.
The content of the invention
In order to solve the deficiencies in the prior art, a kind of general glycoprotein N- sugar chains release side provided by the invention
Method, it is versatile, easy to operate, cost is cheap.
It is an object of the invention to provide a kind of glycoprotein N- sugar chain method for releasing, comprise the following steps：
S1, glycoprotein sample is weighed, be dissolved in concentrated ammonia liquor, 55-70 DEG C of water-bath 12-20h, is obtained in closed container
Reaction solution；
S2, S1 reaction solution is concentrated under reduced pressure drying, adds water to dissolve again, obtains N- sugar chain crude product solutions；
S3, the N- sugar chains crude product solution obtained by S2 is adjusted into pH to neutrality, successively by C18 solid-phase extraction columns and stone
The purifying of black carbon solid-phase extraction column, obtain purified N- sugar chain samples.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, the concentration of the concentrated ammonia liquor is 25-18g/100g.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, after the glycoprotein sample is dissolved in concentrated ammonia liquor, makes
The concentration of glycoprotein sample is 1-20mg/mL.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, as quality >=1g of glycoprotein sample, institute in S2
The step of N- sugar chains crude product solution must pass through savage method combination isoelectric precipitation removing proteins is obtained, then adjusts pH again to neutrality；
As the quality ＜ 1g of glycoprotein sample, gained N- sugar chains crude product solution directly adjusts pH to neutrality in S2, is not required to
The step of passing through savage method combination isoelectric precipitation removing proteins.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, C18 solid-phase extraction column purge processes are：C18 solid phases
Extraction column is first activated with 3 times of column volume acetonitriles, then is balanced with 10 times of column volume distilled waters, then loading, and 10 times of column volumes are double to be steamed
Water elution sugar chain；
Graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with 3 times of column volume acetonitriles, then is used
10 times of column volume distilled waters balance, then by the sample loading through C18 solid-phase extraction columns after purification, first with 10 times of cylinders after loading
Product distilled water elution desalination, is then eluted with 3ml 25ml/100ml acetonitrile solutions, collects eluent, be concentrated under reduced pressure dry
The dry N- sugar chain samples obtained after purification.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, savage methods simultaneously combine isoelectric precipitation removing protein
The step of concrete operations be：First equivalent to 1/5 times N- sugar chain crude product solution volume of addition into N- sugar chain crude product solutions
Savage reagents, mix, then adjust pH to the albumen isoelectric point, centrifuged after stirring 10min, it is standby to collect supernatant；Wherein,
Savage reagents are according to 4 by dichloromethane and n-butanol:1 volume ratio mixes.
Preferably, above-mentioned general glycoprotein N- sugar chain method for releasing, in graphitic carbon solid-phase extraction column purge process, if
N- sugar chains to be purified are acid N- sugar chains, then trifluoroacetic acid is added in eluent acetonitrile solution is made acetonitrile water-trifluoro
Acetic acid solution, the volumetric concentration of trifluoroacetic acid is 0.01% in acetonitrile water-trifluoroacetic acid solution, is then eluted again.
Compared with prior art, general glycoprotein N- sugar chain method for releasing of the invention has the advantages that：
(1) method provided by the invention be based on the N- sugar chains being connected on glycoprotein in concentrated ammonia liquor, amido link (-
CO-NH -) it is broken, generate a kind of unstable osamine.Due to a large amount of excess of ammonia water, therefore osamine in system be present
Protected, so as to reach the purpose of protection sugar chain reducing end, peeling reactions do not occur for the sugar chain for making to be dissociated.When anti-
Ammoniacal liquor is removed after should terminating, and generates reproducibility N- sugar chains, so as to complete the release of glycoprotein N- sugar chains.This method is effectively prevented from
Peeling degradeds with core α -1,3- fucose N- sugar chains.
(2) the N- sugar chains discharged by the present invention exist in the form of reproducibility N- sugar chains, and stability is high, will not drop
Solution, can directly carry out preliminary analysis or derivatization post analysis, available for the release and preparation of different type N- sugar chains, gram
Taken traditional enzyme process and report in the past Chemical releases method the defects of.The method release glycoprotein N- sugar chains of the present invention are simple,
Quickly, it is versatile, cost is cheap, be applicable to neutral N- sugar chains, acid N- sugar chains and core α -1,3- fucose modification
N- sugar chains, and the release efficiency of sugar chain is high, and the micro-analysis and can that can be used for sugar chain is used for the extensive preparation of sugar chain, is
The extensive preparation of N- sugar chains provides feasible method.
Brief description of the drawings
Fig. 1 is the chemical principle of present invention release glycoprotein N- sugar chain methods；
Fig. 2 is the experimental result of the reaction temperature optimization of present invention release glycoprotein N- sugar chain methods；
Fig. 3 is the experimental result of the reaction time optimization of present invention release glycoprotein N- sugar chain methods；
Fig. 4 is the ESI-MS collection of illustrative plates that distinct methods discharge RiboB N- sugar chains；
Wherein, Fig. 4 A are the ESI-MS collection of illustrative plates of PNGaseF enzyme r e lease RiboB N- sugar chains, and Fig. 4 B are that the method for embodiment 1 is released
Put the ESI-MS collection of illustrative plates of RiboB N- sugar chains；
Fig. 5 is the ESI-MS collection of illustrative plates that distinct methods discharge the N- sugar chains on gingko seedses total protein；
Wherein, Fig. 5 A are the ESI-MS collection of illustrative plates of the N- sugar chains on PNGaseF enzyme r e lease gingko seedses total proteins；Fig. 5 B are
The ESI-MS collection of illustrative plates of N- sugar chains on PNGaseA enzyme r e lease gingko seedses total proteins；Fig. 5 C are that the method for embodiment 2 discharges ginkgo kind
The ESI-MS collection of illustrative plates of N- sugar chains on sub- total protein；
When Fig. 6 is N- sugar chains on the gingko seedses total protein of release, molecular weight is the second order mses at 862 (m/z) this peak
Analysis；
Fig. 7 is that MALDI-TOF-MS of the N- sugar chains through GP derivatizations that distinct methods are discharged on enzyme r e lease OVA schemes
Spectrum；
Wherein, Fig. 7 A are MALDI-TOF-MS figure of the N- sugar chains through GP derivatizations on PNGaseF enzyme r e lease OVAs
Spectrum；Fig. 7 B are MALDI-TOF-MS collection of illustrative plates of the N- sugar chains through GP derivatizations on the OVA of sodium hypochlorite release；Fig. 7 C are
MALDI-TOF-MS collection of illustrative plates of the N- sugar chains through GP derivatizations on the OVA of the method for embodiment 3 release；
Fig. 8 is PNGaseF enzymes, every N- sugar of the OVA of three kinds of methods release of sodium hypochlorite and 3 method of embodiment
Chain and block diagram obtained by the relative abundance ratio of internal standard (IS)；
Fig. 9 is the ESI-MS collection of illustrative plates of the release sialylated N- sugar chains of hyclone；
Figure 10 is the ESI-MS collection of illustrative plates of release egg N- sugar chains；
Wherein, in Fig. 4-Fig. 7, Fig. 9 and Figure 10, gray circles represent mannose, and black squares represent N- acetyl grapes
Osamine, black triangle represent fucose, and white five-pointed star represents xylose, and white circle represents galactolipin, and black diamonds represent
Sialic acid.
Embodiment
The embodiment of invention is described in detail below, it is to be understood that protection scope of the present invention not by
The limitation of embodiment.The test method of unreceipted actual conditions in the following example, generally according to normal condition, or
According to the condition proposed by each manufacturer.The embodiments described below, unless otherwise indicated, all temperature units are
Degree Celsius, reaction temperature is room temperature, and room temperature refers to 25 DEG C ± 5 DEG C, and all temperature errors are ± 5 DEG C.
In following embodiments, ribonulease B (RiboB), egg albumin, girard reagent P are purchased from
Sigma-Aldrich companies；PNGase F are purchased from New England BioLabs companies；Lauryl sodium sulfate (SDS), two
Sulphur threitol (DTT), NP-40 are purchased from Aladdin Industrial Inc companies；Hyclone (FBS) is purchased from Thermo
Scientific companies；In being plucked in campus, its total protein extracts gingko seedses for this laboratory；Solid phase extraction column Sep-
Pak C18 (100mg/1mL) are purchased from Waters companies；Solid phase extraction column porous graphitic carbon post (150mg/4mL) is purchased from
Alltech Associates companies；Other reagents are that analysis is pure.Concentrated ammonia liquor is directly bought, and concentration is 26%~28%.It is double
It is that automatically prepared by dual pure water distiller for use for laboratory to steam water；Mass Spectrometric Identification AXIMA ALDI-TOF-MS matter in the present invention
Spectrometer (Japanese Shimadu companies), ESI-MS and multi-stage mses identification (MSn) use electron spray ionisation linear ion hydrazine mass spectrum
(LTQ XL, Thermo Scientific, USA) is detected.
One-level ESI-MS parameter settings are as follows：Sample size, the control of 2 μ L injection annulus；Load sample mobile phase be methanol/water (50%/
50%, v/v)；Flow velocity is 50 μ L/min；Operating voltage is 4kV；Sheath gas is 20arb；Auxiliary gas flow speed is 10arb；Hair
Tubule voltage is 37V；Capillary lens voltage is 250V；Capillary temperature is 300 DEG C；Scan type is one-level full scan；Most
Big injection length is 1000ms；Micro scanning is 3 times；Data acquisition uses LTQ Tune softwares.
Multi-stage mses (MSn) detection parameters are arranged to：Sample size, the control of 2 μ L injection annulus；Load sample mobile phase is volume ratio
50% methanol aqueous solution, flow velocity are 50 μ L/min；Operating voltage is 4kV；Sheath gas is 20arb；Auxiliary gas flow speed is
10arb；Capillary voltage is 37V；Capillary lens voltage is 250V；Capillary temperature is 300 DEG C；Maximum injection length is
1000ms；Micro scanning is 3 times；Collision gas are helium；Isotope width m/z 3.00；Ion collision energy is 35%~
45%；It is 0.25 to activate electric charge；Activationary time is 30ms.
First mass spectrometric (MS) detection and multi-stage mses (MS in the present inventionn) detect, acid N- sugar chains exist in hyclone
Detected under negative ion mode, remaining is detected in the positive-ion mode.
Chinese is as follows corresponding to abbreviation in the present invention：
ACN (acetonitrile), arb arbitrary unit (arbitrary unit, belonging to pressure unit), DMSO (dimethyl sulfoxide (DMSO)),
DTT (dithiothreitol (DTT)), FBS (hyclone), MeOH (methanol), MSn (multi-stage mses), mL (milliliter), min (minute), ms
(millisecond), h (hour), Relative Abundance (relative abundance), SDS (lauryl sodium sulfate), V (volt), M (mol/
ML), IS (internal standard), TFA (trifluoroacetic acid).
Method provided by the invention be based on the N- sugar chains being connected on glycoprotein in concentrated ammonia liquor, amido link (- CO-
NH -) it is broken, generate a kind of unstable osamine.Due to a large amount of excess of ammonia water in system be present, therefore osamine is protected
Shield, so as to reach the purpose of protection sugar chain reducing end, peeling reactions do not occur for the sugar chain for making to be dissociated.When reaction is tied
Ammoniacal liquor is removed after beam, generates reproducibility N- sugar chains, so as to complete the release of glycoprotein N- sugar chains.This method is effectively prevented from carrying
The peeling degradeds of core α -1,3- fucose N- sugar chains.Fig. 1 is the chemical reaction original that Ammonia Process discharges glycoprotein N- sugar chains
Reason.Fig. 2 is the experimental result for the reaction temperature optimization that ammoniacal liquor discharges glycoprotein N- sugar chains；Fig. 3 is that ammoniacal liquor discharges glycoprotein N- sugar
The experimental result of the reaction time optimization of chain；Five lines represent five sugar chains discharged in RiboB albumen, H in Fig. 2 and Fig. 3
Different numerals represents the number of hexose and N-Acetyl-D-glucosamine behind expression hexose, N expression N-Acetyl-D-glucosamines, H and N
Mesh.By, it can be seen that the optimal reaction temperature of N- sugar chains release is 60 DEG C, optimum reacting time is 16h (specific in Fig. 2 and Fig. 3
Method for releasing reference implementation example 1).
Several embodiments are set forth below to illustrate the method for releasing of the present invention：
Embodiment 1
A kind of glycoprotein N- sugar chain method for releasing, releasing object are the neutral N- sugar chains on Ribo B albumen, specific steps
It is as follows：
S1, weighs 5mg Ribo B protein samples, is dissolved in 4ml concentrated ammonia liquors, and the concentration for making Ribo B protein samples is
1.25mg/mL, 60 DEG C of water-bath 16h, obtain reaction solution in closed container；Concentrated ammonia liquor uses commercially available concentration as 25-
28% (i.e. 25-28g/100g) concentrated ammonia liquor；
S2, S1 reaction solution is concentrated under reduced pressure and dries (conventional method, which is concentrated under reduced pressure, to be dried), adds 3mL distilled waters again
Dissolving, obtains N- sugar chain crude product solutions；Due to being the experiment of a small amount of Ribo B protein samples, the step of removing protein can be omitted；
S3, the N- sugar chains crude product solution obtained by S2 is adjusted into pH to neutrality, sample to be purified is obtained, passes through successively
The purifying of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column, purified N- sugar chain samples are obtained, completed in glycoprotein sample
The release of N- sugar chains；
Wherein, C18 solid-phase extraction columns purge process is：C18 solid-phase extraction columns are first activated with 3 times of column volume acetonitriles, then are used
10 times of column volume distilled water balances, sample loading to be purified, 10 times of column volume distilled waters are then eluted into sugar chain.
Graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with 3 times of column volume acetonitriles, then is used
10 times of column volume distilled waters balance, then by the sample loading through C18 solid-phase extraction columns after purification, first with 10 times of cylinders after loading
Product distilled water elution desalination, then, with 3ml 25ml/100ml acetonitrile solutions, (volume fraction of acetonitrile is 25%, and solvent is
Water) eluted, collect eluent, the N- sugar chain samples for being concentrated under reduced pressure after purification.
Reference examples 1
Reference examples 1 using PNGase F enzymolysis release glycoprotein N- sugar chains, releasing object be it is same as Example 1,
It is the neutral N- sugar chains on Ribo B, comprises the following steps that：
5mg Ribo B glycoprotein samples are weighed, are dissolved in 450 μ L distilled waters, add 50 μ L protein denaturation liquid (albumen
The compound method of matter denaturing liquid：50mg SDS and 62mg DTT are dissolved in 1mL distilled waters), in 100 DEG C of heat denatured 10min.Treat
When sample is cooled to room temperature, the 50 μ L enzymolysis buffer solution (compound method of enzymolysis buffer solution is added：It is double that 1.9g sodium phosphates are dissolved in 10mL
Steam in water, with phosphoric acid adjust pH to 7.5), 50 μ L NP-40 (10%, v/v) and 1 μ L PNGase F enzymes, 37 DEG C are reacted 24h.Treat anti-
It should terminate, 100 DEG C of inactivation 5min, sample be dissolved in 1mL distilled waters again after being concentrated under reduced pressure, successively by C18 solid-phase extraction columns
With the purifying of graphitic carbon solid-phase extraction column, purified N- sugar chain samples are obtained, complete the release of N- sugar chains in glycoprotein sample.
Wherein, the purification process of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column is same as Example 1.
The purified N- sugar chain samples that embodiment 1 and reference examples 1 obtain are subjected to ESI-MS detection and analysis respectively, it is different
The ESI-MS collection of illustrative plates of method release RiboB N- sugar chains is as shown in figure 4, wherein, Fig. 4 A are PNGaseF enzyme r e lease RiboB N- sugar
The ESI-MS collection of illustrative plates of chain, Fig. 4 B are the ESI-MS collection of illustrative plates that the method for embodiment 1 (ammoniacal liquor) discharges RiboB N- sugar chains.Both sugar chain kinds
Class is consistent.Show the reliability that the method for embodiment 1 discharges to glycoprotein neutrality N- sugar chains.
Embodiment 2
A kind of glycoprotein N- sugar chain method for releasing, releasing object are N- sugar chains on gingko seedses total protein, it is known that ginkgo kind
N- sugar chains containing the modification of core α -1,3- fucose on sub- total protein, are comprised the following steps that：
S1, weighs 10mg gingko seedses total proteins, is dissolved in 4ml concentrated ammonia liquors, and the concentration for making gingko seedses total protein is
2.5mg/mL, 60 DEG C of water-bath 16h, obtain reaction solution in closed container；Concentrated ammonia liquor uses commercially available concentration as 25-28%
(unit is 25-28g/100g) concentrated ammonia liquor；
S2, S1 reaction solution are concentrated under reduced pressure drying, add 3mL distilled waters to dissolve again, obtain N- sugar chain crude product solutions；Due to being
A small amount of gingko seedses total protein samples experiments, the step of removing protein can be omitted；
S3, the N- sugar chains crude product solution obtained by S2 is adjusted into pH to neutrality, sample to be purified is obtained, passes through successively
The purifying of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column, purified N- sugar chain samples are obtained, completed in glycoprotein sample
The release and purifying of N- sugar chains；
Wherein, C18 solid-phase extraction columns purge process is：C18 solid-phase extraction columns are first activated with 3 times of column volume acetonitriles, then are used
15 times of column volume distilled water balances, sample loading to be purified, 15 times of column volume distilled waters are then eluted into sugar chain.
Graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with 3 times of column volume acetonitriles, then is used
15 times of column volume distilled waters balance, then by the sample loading through C18 solid-phase extraction columns after purification, first with 15 times of cylinders after loading
Product distilled water elution desalination, then, with 3ml 25ml/100ml acetonitrile solutions, (volume fraction of acetonitrile is 25%, and solvent is
Water) eluted, collect eluent, the N- sugar chain samples for being concentrated under reduced pressure after purification.
Reference examples 2-1
Reference examples 2-1 using PNGase F enzymolysis release glycoprotein N- sugar chains, releasing object be it is same as Example 2,
It is N- sugar chains on gingko seedses total protein, comprises the following steps that：
5mg gingko seedses total protein samples are weighed, are dissolved in 450 μ L distilled waters, add 50 μ L protein denaturation liquid (albumen
The compound method of matter denaturing liquid：50mg SDS and 62mg DTT are dissolved in 1mL distilled waters), in 100 DEG C of heat denatured 10min.Treat
When sample is cooled to room temperature, the 50 μ L enzymolysis buffer solution (compound method of enzymolysis buffer solution is added：It is double that 1.9g sodium phosphates are dissolved in 10mL
Steam in water, with phosphoric acid adjust pH to 7.5), 50 μ L NP-40 (10%, v/v) and 1 μ L PNGase F enzymes, 37 DEG C are reacted 24h.Treat anti-
It should terminate, 100 DEG C of inactivation 5min, sample be dissolved in 1mL distilled waters again after being concentrated under reduced pressure, successively by C18 solid-phase extraction columns
With the purifying of graphitic carbon solid-phase extraction column, purified N- sugar chain samples are obtained, complete the release of N- sugar chains in glycoprotein sample.
Wherein, the purification process of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column is same as Example 2.
Reference examples 2-2
Reference examples 2-2 using PNGase A enzymolysis release glycoprotein N- sugar chains, releasing object be it is same as Example 2,
It is N- sugar chains on gingko seedses total protein, comprises the following steps that：
5mg gingko seedses total protein samples are weighed, are dissolved in the hydrochloride buffer (pH=that 1.25ml contains 3mg pepsins
2) in, 16h, 100 DEG C of xeothermic 5min after reaction terminates are reacted in 37 DEG C.Sample is dried up with nitrogen, adds 1mL lemon acid bufferings
Liquid (0.1mol/L, pH=5) and 1.25 μ L PNGase A, react 48h under the conditions of 37 DEG C, after reaction terminates 100 DEG C it is xeothermic
5min, it is then centrifuged for, takes supernatant concentration drying.Sample is dissolved in 1mL distilled waters again, is then first purified with C18 pillars, then use stone
Black carbon post purifying.Purification process method is with embodiment 2, the N- sugar chain samples purified, -20 DEG C of preservations after drying.
The obtained purified N- sugar chain samples of embodiment 2, reference examples 2-1 reference examples 2-2 are subjected to ESI-MS inspections respectively
Analysis is surveyed, distinct methods discharge the ESI-MS collection of illustrative plates of the N- sugar chains on gingko seedses total protein as shown in figure 5, wherein, Fig. 5 A are
The ESI-MS collection of illustrative plates of N- sugar chains on PNGaseF enzyme r e lease gingko seedses total proteins, without α containing core -1,3- fucoses
The N- sugar chains of modification；Fig. 5 B are the ESI-MS collection of illustrative plates of the N- sugar chains on PNGaseA enzyme r e lease gingko seedses total proteins, are contained wherein having
The N- sugar chains of core α -1,3- fucoses modification；Fig. 5 C are the N- on the method for embodiment 2 (ammoniacal liquor) release gingko seedses total protein
The ESI-MS collection of illustrative plates of sugar chain, wherein having the species and PNGaseA enzyme r e leases sugar of the N- sugar chains of α containing core -1,3- fucoses modification
Chain species is consistent；As a result show that the method for embodiment 2 discharges to the N- sugar chains that -1,3- of α containing core fucoses in glycoprotein are modified
Reliability.
Embodiment 2 (Fig. 5 C), compared with PNGaseA enzyme r e leases (Fig. 5 B) collection of illustrative plates, wherein having more a molecular weight as 862
(m/z) mass spectrum, we have carried out second mass analysis to it, are as a result illustrated in figure 6 the second order mses figure at the peak.Fig. 6 is
On the gingko seedses total protein of release during N- sugar chains, molecular weight is the second mass analysis result at 862 (m/z) this peak.The knot
Fruit proves that this peak may be occurred by N- sugar chains (molecular weight 1211, m/z) small part of α containing core -1,3- fucoses modification
Peeling degrades, and this result illustrates to have small part hair during the N- sugar chains of ammoniacal liquor release core α -1,3- fucose modification
Raw peeling degradeds, but we pass through practical proof, and the palliating degradation degree is very small, does not influence the content of N- sugar chains, content completely
Error does not influence it and prepares result within 0.2%.
Embodiment 3
A kind of glycoprotein N- sugar chain method for releasing, releasing object are OVA N- sugar chains (neutral N- sugar chains), specific step
Suddenly with embodiment 1, difference is, protein sample is replaced with into 5mg OVAs.After the N- sugar chain samples purified add β-
Cyclodextrin makees internal standard (IS), and MALDI-TOF-MS mass spectral analyses are carried out after target plate derivatization marks GP.
Reference examples 3-1
Reference examples 3-1 using PNGase F enzymolysis release glycoprotein N- sugar chains, releasing object be it is same as Example 3,
It is OVA N- sugar chains, comprises the following steps that：
5mg OVA protein samples are weighed, are dissolved in 450 μ L distilled waters, add 50 μ L protein denaturation liquid (protein
The compound method of denaturing liquid：50mg SDS and 62mg DTT are dissolved in 1mL distilled waters), in 100 DEG C of heat denatured 10min.Treat sample
When product are cooled to room temperature, the 50 μ L enzymolysis buffer solution (compound method of enzymolysis buffer solution is added：1.9g sodium phosphates are dissolved in the double steamings of 10mL
In water, with phosphoric acid adjust pH to 7.5), 50 μ L NP-40 (10%, v/v) and 1 μ L PNGase F enzymes, 37 DEG C are reacted 24h.Question response
Terminate, 100 DEG C of inactivation 5min, sample be dissolved in 1mL distilled waters again after being concentrated under reduced pressure, successively by C18 solid-phase extraction columns and
The purifying of graphitic carbon solid-phase extraction column, purified N- sugar chain samples are obtained, complete the release of N- sugar chains in glycoprotein sample.Its
In, the purification process of C18 solid-phase extraction columns and graphitic carbon solid-phase extraction column is same as Example 3.The N- sugar chain samples purified
Beta-schardinger dextrin is added after product and makees internal standard (IS), MALDI-TOF-MS mass spectral analyses are carried out after target plate derivatization marks GP.
Reference examples 3-2
Reference examples 3-2's discharges glycoprotein N- sugar chains using sodium hypochlorite, and releasing object is same as Example 3, is
OVA N- sugar chains, are comprised the following steps that：
5mg OVAs are weighed, are dissolved in 250 μ L distilled waters, add 50 μ L 6% sodium hypochlorite, vibrate 15min
Afterwards, 2.5 μ L formic acid is added, vibrates 5min, 10000rpm centrifugation 10min, reject precipitation.Weight is molten after supernatant is concentrated under reduced pressure
In 500 μ L distilled waters, C18 solid phase extraction columns and graphitic carbon solid phase extraction column purifying N- sugar chains are crossed respectively, wherein, C18
The purification process of solid-phase extraction column and graphitic carbon solid-phase extraction column is same as Example 3, adds after the N- sugar chain samples purified
Enter beta-schardinger dextrin and make internal standard (IS), MALDI-TOF-MS mass spectral analyses are carried out after target plate derivatization marks GP.
The MALDI-TOF-MS mass spectral analyses of embodiment 3, reference examples 3-1, reference examples 3-2 are as shown in fig. 7, Fig. 7 is difference
MALDI-TOF-MS collection of illustrative plates of the N- sugar chains through GP derivatizations on method release enzyme r e lease OVA；
Wherein, Fig. 7 A are MALDI-TOF-MS figure of the N- sugar chains through GP derivatizations on PNGaseF enzyme r e lease OVAs
Spectrum；Fig. 7 B are MALDI-TOF-MS collection of illustrative plates of the N- sugar chains through GP derivatizations on the OVA of sodium hypochlorite release；Fig. 7 C are
MALDI-TOF-MS collection of illustrative plates of the N- sugar chains through GP derivatizations on the OVA of the method for embodiment 3 (ammoniacal liquor) release；With every
The relative abundance of N- sugar chains obtains relative abundance ratio than interior target relative abundance, and this ratio is carried out to analyze to obtain knot shown in Fig. 8
Fruit, Fig. 8 be PNGaseF enzymes, the three kinds of methods release of sodium hypochlorite and 3 method of embodiment OVA every N- sugar chain with it is interior
Mark block diagram obtained by the relative abundance ratio of (IS).As a result show, from the point of view of most of N- sugar chains, the method release of embodiment 3
The efficiency of N- sugar chains is higher than PNGaseF enzymes and the release efficiency of sodium hypochlorite.
Embodiment 4
A kind of glycoprotein N- sugar chain method for releasing, releasing object are sialylated N- sugar chains, specific steps in hyclone
With embodiment 1, difference is, protein sample is replaced with into 10mg hyclone protein samples, the dosage of concentrated ammonia liquor is changed to 8ml.
ESI-MS mass spectral analyses are carried out after to the N- sugar chain samples of purifying.
Embodiment 5
A kind of glycoprotein N- sugar chain method for releasing, releasing object be or egg in N- sugar chains, specific steps are the same as real
Example 1 is applied, difference is, protein sample is replaced with into 10mg egg freeze-dried powder samples, the dosage of concentrated ammonia liquor is changed to 8ml.
Fig. 9 is that the method for embodiment 4 discharges the ESI-MS collection of illustrative plates of the sialylated N- sugar chains of hyclone；Figure 10 is embodiment
The ESI-MS collection of illustrative plates of 5 method release egg N- sugar chains.The two detectable N- sugar chain, further proves to release using ammoniacal liquor
Feasibility, the versatility of albumen N- sugar chains with sugar.
Embodiment 6
A kind of glycoprotein N- sugar chain method for releasing, releasing object are sialylated N- sugar chains, specific steps in hyclone
It is as follows：
S1, weighs 1g hyclone protein samples, is dissolved in 1000ml concentrated ammonia liquors, and the concentration for making hyclone albumen is
1mg/mL, 55 DEG C of water-bath 12h, obtain reaction solution in closed container；Be concentrated under reduced pressure drying, adds distilled water weight molten；
S2, S1 lysate savage method combination isoelectric precipitation removing proteins, 10000rpm centrifugation 10min, are removed big
The protein and peptide of amount, it is concentrated under reduced pressure and dries to remove organic solvent, add distilled water to dissolve again, obtain N- sugar chain crude product solutions；
Wherein, savage methods and with reference to isoelectric precipitation removing protein concrete operations it is：First dissolved to after being concentrated under reduced pressure with water
Solution in plus 1/5 times of volume savage reagents, mix, then adjust pH to the albumen isoelectric point, centrifuged after stirring 10min,
It is standby to collect supernatant, you can remove substantial amounts of protein and peptide；Wherein, savage reagents be by dichloromethane and n-butanol by
According to 4:1 volume ratio mixes.
For S3, S3 operating procedure with embodiment 1, difference is that eluent uses the acetonitrile water of 3ml 25%-trifluoroacetic acid solution
(volume fraction of acetonitrile is 25%, and the volume fraction of trifluoroacetic acid is 1%, and solvent is water).
Embodiment 7
A kind of glycoprotein N- sugar chain method for releasing, releasing object are sialylated N- sugar chains, specific steps in hyclone
It is as follows：
S1, weighs 20mg hyclone protein samples, is dissolved in 1ml concentrated ammonia liquors, and the concentration for making hyclone albumen is
20mg/mL, 70 DEG C of water-bath 20h, obtain reaction solution in closed container；
S2, S1 reaction solution are concentrated under reduced pressure drying, add 3mL distilled waters to dissolve again, obtain N- sugar chain crude product solutions；Due to being
A small amount of Ribo B protein samples experiments, the step of removing protein can be omitted；
S3, S3 operating procedure are the same as embodiment 1.
It should be noted that when being related to number range in claims of the present invention, it is thus understood that each number range
Any one numerical value can be selected between two end points and two end points, because the step method of use is identical with embodiment,
In order to prevent repeating, the present invention describes preferred embodiment and its effect, but those skilled in the art once know base
This creative concept, then other change and modification can be made to these embodiments.So appended claims are intended to be construed to
Including preferred embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without departing from the present invention to the present invention
God and scope.So, if these modifications and variations of the present invention belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these changes and modification.
Claims (7)
1. a kind of general glycoprotein N- sugar chain method for releasing, it is characterised in that comprise the following steps：
S1, glycoprotein sample is weighed, be dissolved in concentrated ammonia liquor, 55-70 DEG C of water-bath 12-20h, is reacted in closed container
Liquid；
S2, S1 reaction solution is concentrated under reduced pressure drying, adds water to dissolve again, obtains N- sugar chain crude product solutions；
S3, the N- sugar chains crude product solution obtained by S2 is adjusted into pH to neutrality, successively by C18 solid-phase extraction columns and graphitic carbon
The purifying of solid-phase extraction column, obtain purified N- sugar chain samples.
2. general glycoprotein N- sugar chain method for releasing according to claim 1, it is characterised in that the concentrated ammonia liquor it is dense
Spend for 25-28g/100g.
3. general glycoprotein N- sugar chain method for releasing according to claim 2, it is characterised in that the glycoprotein sample
After being dissolved in concentrated ammonia liquor, the concentration for making glycoprotein sample is 1-20mg/mL.
4. general glycoprotein N- sugar chain method for releasing according to claim 1, it is characterised in that when glycoprotein sample
During quality >=1g, gained N- sugar chains crude product solution the step of must passing through savage method combination isoelectric precipitation removing proteins in S2, so
PH is adjusted again afterwards to neutrality；
As the quality ＜ 1g of glycoprotein sample, gained N- sugar chains crude product solution directly adjusts pH to neutrality in S2, it is not necessary to passes through
The step of crossing savage method combination isoelectric precipitation removing proteins.
5. general glycoprotein N- sugar chain method for releasing according to claim 1, it is characterised in that C18 solid-phase extraction columns
Purge process is：C18 solid-phase extraction columns are first activated with 3 times of column volume acetonitriles, then are balanced with 10 times of column volume distilled waters, Ran Houshang
Sample, 10 times of column volume distilled waters elute sugar chain；
Graphitic carbon solid-phase extraction column purge process is：Graphitic carbon solid-phase extraction column is first activated with 3 times of column volume acetonitriles, then with 10 times
Column volume distilled water balances, then by the sample loading through C18 solid-phase extraction columns after purification, first with 10 times of column volumes pair after loading
Water elution desalination is steamed, is then eluted with 3ml 25ml/100ml acetonitrile solutions, collects eluent, is concentrated under reduced pressure dry
N- sugar chain samples after purification.
6. general glycoprotein N- sugar chain method for releasing according to claim 4, it is characterised in that savage methods simultaneously combine
The concrete operations of the step of isoelectric precipitation removing protein are：Equivalent to 1/5 times N- sugar chain is first added into N- sugar chain crude product solutions
The savage reagents of crude product solution volume, mix, then adjust pH to the albumen isoelectric point, centrifuged after stirring 10min, collect supernatant
Liquid is standby；Wherein, savage reagents are according to 4 by dichloromethane and n-butanol:1 volume ratio mixes.
7. general glycoprotein N- sugar chain method for releasing according to claim 5, it is characterised in that graphitic carbon SPE
In post purge process, if N- sugar chains to be purified are acid N- sugar chains, trifluoro second is added in eluent acetonitrile solution
Acid is made acetonitrile water-trifluoroacetic acid solution, and the volumetric concentration of trifluoroacetic acid is 0.01% in acetonitrile water-trifluoroacetic acid solution, so
Eluted again afterwards.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710984428.8A CN107793496A (en) | 2017-10-20 | 2017-10-20 | A kind of general glycoprotein N sugar chain method for releasing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710984428.8A CN107793496A (en) | 2017-10-20 | 2017-10-20 | A kind of general glycoprotein N sugar chain method for releasing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107793496A true CN107793496A (en) | 2018-03-13 |
Family
ID=61532719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710984428.8A Pending CN107793496A (en) | 2017-10-20 | 2017-10-20 | A kind of general glycoprotein N sugar chain method for releasing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107793496A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633066A (en) * | 2019-01-10 | 2019-04-16 | 四川大学华西医院 | A kind of inexpensive, simple and quick glycoprotein N- sugar chain analysis method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010071817A3 (en) * | 2008-12-19 | 2010-11-18 | Momenta Pharmaceuticals, Inc. | Characterization of o-linked glycans |
| CN104974994A (en) * | 2014-04-02 | 2015-10-14 | 复旦大学 | Glycosidase for deglycosylation of N-glycoproteins and application thereof |
-
2017
- 2017-10-20 CN CN201710984428.8A patent/CN107793496A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010071817A3 (en) * | 2008-12-19 | 2010-11-18 | Momenta Pharmaceuticals, Inc. | Characterization of o-linked glycans |
| CN104974994A (en) * | 2014-04-02 | 2015-10-14 | 复旦大学 | Glycosidase for deglycosylation of N-glycoproteins and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| YUNPING HUANG ET AL.: "Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis", 《ANALYTICAL CHEMISTRY》 * |
| 孙伟: "枸杞糖蛋Lbgp5-B的分离纯化及其N－糖链的结构解析", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 张真庆: "质谱分析外源性和内源性寡糖的方法学研究", 《中国优秀博硕士学位论文全文数据库 (博士) 农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633066A (en) * | 2019-01-10 | 2019-04-16 | 四川大学华西医院 | A kind of inexpensive, simple and quick glycoprotein N- sugar chain analysis method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2013628B1 (en) | Methods of evaluating peptide mixtures | |
| Weeds et al. | The amino‐acid sequence of the alkali light chains of rabbit skeletal‐muscle myosin | |
| Poulsen | Direct screening of a dynamic combinatorial library using mass spectrometry | |
| CN104345107B (en) | Lac Bovis seu Bubali serum albumin immue quantitative detection reagent box in a kind of breast or milk product | |
| CN107907620A (en) | The method that ultrasonic extraction Solid Phase Extraction pre-treatment combination LC-MS technology detects six 24 kinds of antibiotic of class in feces of livestock and poultry at the same time | |
| CN104020216B (en) | Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner | |
| CN106093244A (en) | A kind of sheep characteristic polypeptide and application thereof | |
| Esteban Warren et al. | Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination | |
| Zhang et al. | PNGase F-mediated incorporation of 18 O into glycans for relative glycan quantitation | |
| CN106918636A (en) | The authentication method of protein group and its application | |
| Martino et al. | Chemical synthesis of cross‐linked poly (KGGVG), an elastin‐like biopolymer | |
| CN107793496A (en) | A kind of general glycoprotein N sugar chain method for releasing | |
| Barrientos et al. | Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP | |
| CN104931308B (en) | A kind of method for preparing fumonisin B1, B2 and B3 standard items simultaneously | |
| Xu et al. | Rapid sensing and imaging of methylglyoxal in living cells enabled by a near-infrared fluorescent probe | |
| CN106053697B (en) | A kind of ox source property characteristic polypeptide and its application | |
| CN107389805A (en) | Reproducibility discharges glycoprotein N sugar chains and its derivative separation and the method analyzed and identified | |
| Wu et al. | An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose | |
| Friess et al. | Mass spectrometric noncovalent probing of amino acids in peptides and proteins | |
| Bonomo et al. | Chiral recognition by the copper (II) complex of 6‐deoxy‐6‐N‐(2‐methylaminopyridine)‐β‐cyclodextrin | |
| CN106198790A (en) | A kind of horse and mule common characteristic polypeptide and application thereof | |
| Scholten et al. | Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9: Im9 bacteriotoxin—immunity protein complex | |
| CN102866226B (en) | Method for using pyrazosulfuron-ethyl molecularly imprinted polymer for analyzing sulfonylurea herbicide and application thereof | |
| CN112362798A (en) | Detection method of cannabidiol in cosmetics | |
| CN103278586A (en) | Extracting and detecting method for dicyandiamide component in dairy products |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180313 |