CN109738533A - A kind of enrichment of high throughput Simple cell O- glycosylation site, identification method - Google Patents
A kind of enrichment of high throughput Simple cell O- glycosylation site, identification method Download PDFInfo
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- CN109738533A CN109738533A CN201811650406.9A CN201811650406A CN109738533A CN 109738533 A CN109738533 A CN 109738533A CN 201811650406 A CN201811650406 A CN 201811650406A CN 109738533 A CN109738533 A CN 109738533A
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- agglutinin
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Abstract
The invention belongs to protein analysis technical field, specially a kind of enrichment of high-throughput Simple cell O- glycosylation site, identification method.The method of the present invention includes: so that the synthesis of O-GalNAc sugar chain is inhibited the level in single GalNAc by the way that O-GalNAc sugar chain synthetic inhibitor is added in cell culture environment；Then it assists agglutinin beneficiation technologies to realize the enrichment of O-GalNAc glycopeptide by ultrafiltration, the O- glycopeptide being enriched to is discharged using monosaccharide competition mechanism；Finally by LC-MS, high throughput identification O-GalNAc glycosylation site.The present invention solves the problems, such as because O- sugar chain discharges the missing of enzyme and without the site the reasons bring O-GalNAc such as conservative decorating site sequence high throughput identification difficulty, simplify O- sugar chain strategy, the ultrafiltration auxiliary affine beneficiation technologies of agglutinin by inhibitor, realizes the Large scale identification in the site O-GalNAc.
Description
Technical field
The invention belongs to protein analysis technical fields, and in particular to a kind of high throughput Simple cell O- glycosylation site
Enrichment, identification method.
Background technique
Glycosylation is most common rear modification in protein modification.At present N- glycosylation modified analytical technology increasingly at
Ripe, but O- is glycosylation modified, especially in O-GalNAc(O- acetylgalactosamine) analysis of modification still has very big bottleneck.It is high
The missing and non-conservative decorating site sequence signature (such as NXS/T motif of N sugar) for imitating special O- sugar chain release enzyme make
The existing technology of N- glycosylation cannot directly be continued to use by obtaining the research of the site O-GalNAc.At the same time, itself high molecular weight of sugar chain,
The characteristic of glycosidic bond easy fracture etc. causes O-GalNAc mass spectrogram to search library cannot be with acetylation, phosphorylation and O-GlcNAc etc.
Modification is realized by the way that the modification of fixed member amount is arranged during searching library like that.These all limit O-GalNAc glycosylation and repair
Adorn the Large scale identification in site.The tactful gene means of the simple cell (Simple cell) of Clausen et al. development in recent years are struck
Except O- sugar originates the chaperone COSMC of extension enzyme, the sugar chain structure of O-GalNAc is simplified, the O- for generating cell
The sugar chain of GalNAc is all reduced to the form of only one monosaccharide O-GalNAc, then is identified, current maximum thereby is achieved
The site O-GalNAc data set.However, this method, which needs to stablize, knocks out chaperone gene, from experimental implementation technical difficulty
Height limits the application and popularization of this method to a certain extent.Therefore, the present invention is using a kind of " chemistry simplifies cell strategy "
Realize the extensive enrichment and identification of O- glycosylation site, this method, which synthesizes O-GalNAc sugar chain especially by inhibitor, to be inhibited
In the level of single GalNAc, realize that the high-throughput of O-GalNAc is enriched with further combined with the affine enrichment of ultrafiltration auxiliary agglutinin,
Then by the mass spectrographic analysis of LC-MS, high-throughput efficient O- glycosylation site identification is realized.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of high passes simple, easy to operate, rapidly and efficiently
Measure enrichment, the identification method of Simple cell O- glycosylation site.
The enrichment of high throughput Simple cell O- glycosylation site provided by the invention, identification method, the specific steps are as follows:
(1) by the way that O-GalNAc sugar chain synthetic inhibitor is added in cell culture environment, inhibit the synthesis of O-GalNAc sugar chain
In the level of single GalNAc；
(2) it then assists agglutinin beneficiation technologies to realize the enrichment of O-GalNAc glycopeptide by ultrafiltration, utilizes monosaccharide competition mechanism
Discharge the O- glycopeptide being enriched to；
(3) finally by LC-MS, high throughput identification O-GalNAc glycosylation site.
The enrichment of high throughput Simple cell O- glycosylation site provided by the invention, identification method, further specific behaviour
It is as follows to make process:
(1) O- sugar chain synthetic inhibitor is added in cell culture medium；
(2) cell is cultivated in the culture medium containing inhibitor；
(3) culture medium is discarded, conventional cell collection, protein cleavage and pancreatin enzymatic hydrolysis are carried out after cleaning 3 times with PBS；
(4) PNGase F and sialidase removal N- sugar chain and sialic acid is added；
(5) peptide hydrolysis desalination is then lyophilized with C18 pillar；
(6) agglutinin and the peptide fragment sample after freeze-drying are incubated with；
(7) the agglutinin solution after incubation is transferred to super filter tube, carries out ultrafiltration, and clean 5 times or more, wash away not with agglutinin
In conjunction with non-saccharide egg peptide；
(8) GalNAc of 0.1 ~ 1mol/L of final concentration is added in system, is incubated for 0.5 ~ 2h, it, will be with by monosaccharide Competition
The O- glycopeptide that agglutinin combines releases；
(9) ultrafiltration, and clean 2 ~ 5 times, merge the O- glycopeptide of collection and the mixed solution of GalNAc；
(10) it by the salt and GalNAc in C18 pillar desalination removing system, is then lyophilized；
(11) LC-MS analysis is done, identifies O-GalNAc glycosylation site.
In the present invention, used O- sugar chain synthetic inhibitor includes but is not limited to benzyl 2- acetylamino 2- deoxidation α-D
Galactoside (Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside).
In process (1) of the present invention, used culture medium is the culture medium for the recommendation that the cell buys source；Step (1)
The middle concentration range that inhibitor is added is in 1 ~ 10 mM.
In process (2) of the present invention, the time that the cell is cultivated under inhibitor environment is 24 ~ 48h.
In process (4) of the present invention, the PNGase F and sialidase of addition are with the corresponding 1 mg starting albumen of 300 ~ 500 U
Ratio is added, and 37 DEG C are incubated for 14 ~ 18 hours.
In process (6) of the present invention, the agglutinin of addition is vicia villosa lectin (Viciavillosa
Lectin, VVL), the concentration of addition be 1 ~ 5 microgram/microlitre；Incubation at room temperature, incubation time are 0.5 ~ 3 h, are incubated for and super
The solution of filter is that 10 ~ 100 mMTris/HCl pH are 7 ~ 8,0 ~ 2mM MnCl2, 0~2mM CaCl2, 0~2 MNaCl。
Inhibitor is added in cell culture in the present invention, so that the extension of O-GalNAc sugar chain all in cell is pressed down
System, all O-GalNAc sugar chains exist in the form of single GalNAc, simplify the structure of sugar chain, then remove saliva that may be present
After liquid acid, such O- sugar can be identified by agglutinin VVL.It, can be with fast high-flux by ultrafiltration ancillary technique and C18 desalination technology
The enrichment of ground realization O- sugar.The present invention have it is easy to operate, quickly, it is high-throughput the features such as.
Detailed description of the invention
Fig. 1 is the flow chart of this method.
Fig. 2 is the result figure of agglutinin trace.
Specific embodiment
The method of the present invention is further described in detail below by specific embodiment.
Embodiment 1:
The agglutinin trace that the albumen extracted before and after inhibitor is incubated for VVL and corresponding avidin is added in Hela cell
It is separately added into 2 mM, 5 mM, 10 mM inhibitor in DMEM culture medium and carries out egg after 48h respectively for 24 hours with cell incubation
White extraction carries out agglutinin trace again.
Steps are as follows for agglutinin agglutination:
(1) electrophoresis: polyacrylamide gel electrophoresis glue is prepared first.After preparing gel, take 20 μ g sample solutions that loading is added
Buffer is suspended, and is added in swimming lane after centrifugation.Separately plus marker is compareed as molecular weight.Electrophoresis plate is put into electrophoresis tank, if
Set electric current: electric current is set as 10 mA/ glue when concentration glue separation, and when bromophenol blue forward position reaches separation gel interface, electric current is adjusted to 20
MA/ glue.Until stopping when bromophenol blue Front distance glue bottom 0.5cm or so；
(2) transferring film is closed, and is incubated for 3 μ g/mL primary antibodies (agglutinin lectin VVL) and each 30 points of 0.8 μ g/mL secondary antibody (avidin)
Clock post-exposure analysis.
Fig. 2 is the result figure of agglutinin trace.
It is dramatically increased as can be seen that simplifying sugar-type after inhibitor is added, illustrates that inhibitor can reach expected and inhibit sugar
Chain elongation, the effect for generating list GalNAc sugar-type glycoprotein；Simultaneously as can be seen that as the time increases the increasing with inhibitor concentration
Add, generates and simplify the increase of sugar-type protein content.10 mM Benzyl 2-acetamido-2- are added in cell cultivation process
It is best to continue 48 hours effects made of culture by deoxy-α-D-galactopyranoside.O- after inhibiting, in cell
GalNAc sugar-type can significantly simplify, and by agglutinin, can be effectively enriched with to such glycoprotein.In addition, natural O-
For GalNAc without fixed composition form, there is presently no softwares can be parsed on a large scale to its mass spectrometric data, simplifies sugar chain
Strategy, conventional interpretation of mass spectra software can analyze O-GalNAc glycoprotein.
Embodiment 2
The site Hela cell O-GalNAc Large scale identification:
10 mMBenzyl 2-acetamido-2-deoxy-α-D-galactopyranoside will be added in DMEM culture medium
Inhibitor is cleaned 3 times with PBS after being incubated for 48h in cell, then carries out protein cleavage, proteolysis.By corresponding 1 mg of 500 U
The ratio for originating albumen is added PNGase F and sialidase, 37 DEG C be incubated for 16 hours after use C18 pillar desalination, freeze-drying.Then
It is added 200 microlitres of VVL and buffer and VVL agglutinin in the peptide fragment of freeze-drying, the concentration of agglutinin is 2.5 micrograms/micro-
It rises.Be incubated at room temperature 1h, be transferred to super filter tube, 12000 g are centrifuged 15 minutes, be then added 200 microlitres of buffer, 12000 g from
It the heart 15 minutes, cleans 5 times.The GalNAc of final concentration of 1 M is then added, 12000g is centrifuged 15 minutes after 1.5 h of shaken at room temperature,
And twice with 100 microlitres of buffer solution for cleaning, merge the solution that 3 ultrafiltration are got off, and with C18 pillar desalination, liquor charging matter after freeze-drying
It is combined mass spectral analysis.The glycoprotein of 215 O-GalNAc modification, 224 glycopeptide segments and 498 corresponding are identified altogether
Site.This method can quickly and easily realize that the site O-GalNAc is enriched with and quantitative method on a large scale, has filled up existing side
The blank of method.
Claims (7)
1. a kind of enrichment of high throughput Simple cell O- glycosylation site, identification method, which is characterized in that specific step is as follows:
(1) by the way that O-GalNAc sugar chain synthetic inhibitor is added in cell culture environment, inhibit the synthesis of O-GalNAc sugar chain
In the level of single GalNAc；
(2) it then assists agglutinin beneficiation technologies to realize the enrichment of O-GalNAc glycopeptide by ultrafiltration, utilizes monosaccharide competition mechanism
Discharge the O- glycopeptide being enriched to；
(3) finally by LC-MS, high throughput identification O-GalNAc glycosylation site.
2. the method according to claim 1, wherein concrete operations process are as follows:
(1) O- sugar chain synthetic inhibitor is added in cell culture medium；
(2) cell is cultivated in the culture medium containing inhibitor；
(3) culture medium is discarded, conventional cell collection, protein cleavage and pancreatin enzymatic hydrolysis are carried out after cleaning 3 times with PBS；
(4) PNGase F and sialidase removal N- sugar chain and sialic acid is added；
(5) the peptide fragment desalination after enzymatic hydrolysis is then lyophilized with C18 pillar；
(6) agglutinin and the peptide fragment after desalination are incubated with；
(7) the agglutinin solution after incubation is transferred to super filter tube, carries out ultrafiltration, and clean 5 times or more, wash away not with agglutinin
In conjunction with non-saccharide egg peptide；
(8) GalNAc of 0.1 ~ 1mol/L of final concentration is added in system, is incubated for 0.5 ~ 2 h, it, will be with by monosaccharide Competition
The O- glycopeptide that agglutinin combines releases；
(9) ultrafiltration, and clean 2 ~ 5 times, merge the O- glycopeptide of collection and the mixed solution of GalNAc；
(10) it by the salt and GalNAc in C18 pillar desalination removing system, is then lyophilized；
(11) LC-MS analysis is done, identifies O-GalNAc glycosylation site.
3. method according to claim 1 or 2, which is characterized in that used O- sugar chain synthetic inhibitor is Benzyl
2-acetamido-2-deoxy–α -D-galactopyranoside。
4. method according to claim 1 or 2, which is characterized in that the concentration that inhibitor is added in process (1) is 1 ~ 10
mM。
5. method according to claim 1 or 2, which is characterized in that in process (2), the cell is under inhibitor environment
The time of culture is 24 ~ 48h.
6. method according to claim 1 or 2, which is characterized in that in process (4), the PNGase F and sialic acid of addition
Enzyme is added with the ratio of the corresponding 1 mg starting albumen of 300 ~ 500 U, and 37 DEG C are incubated for 14 ~ 18 hours.
7. method according to claim 1 or 2, which is characterized in that in process (6), the agglutinin of addition is long pubescence
Vetch lectin, the concentration of addition be 1 ~ 5 microgram/microlitre；Incubation at room temperature, incubation time be 0.5 ~ 3 h, be incubated for
And it is 7 ~ 8,0 ~ 2mM MnCl that the solution of ultrafiltration, which is 10 ~ 100 mMTris/HCl pH,2, 0~2mM CaCl2, 0~2 MNaCl。
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Cited By (1)
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| CN114252629A (en) * | 2021-11-25 | 2022-03-29 | 苏州大学 | Analysis method based on solid-phase glycoprotein enrichment and Tn glycopeptide enzyme digestion and application |
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