CN109738533B

CN109738533B - High-throughput simple cell O-glycosylation site enrichment and identification method

Info

Publication number: CN109738533B
Application number: CN201811650406.9A
Authority: CN
Inventors: 黄江铭; 曹纬倩; 蒋碧云; 杨芃原
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-12-31
Filing date: 2018-12-31
Publication date: 2022-06-21
Anticipated expiration: 2038-12-31
Also published as: CN109738533A

Abstract

The invention belongs to the technical field of protein analysis, and particularly relates to a high-throughput simple method for enriching and identifying O-glycosylation sites of cells. The method comprises the following steps: suppressing O-GalNAc sugar chain synthesis at a level of a single GalNAc by adding an inhibitor of O-GalNAc sugar chain synthesis in a cell culture environment; then the enrichment of O-GalNAc glycopeptide is realized by an ultrafiltration auxiliary lectin enrichment technology, and the enriched O-glycopeptide is released by utilizing a monosaccharide competition mechanism; and finally, identifying the O-GalNAc glycosylation site through LC-MS in a high-throughput manner. The invention solves the problem of difficult high-throughput identification of the O-GalNAc locus caused by the reasons of deletion of O-sugar chain releaser, no conservative modified site sequence and the like, and realizes the large-scale identification of the O-GalNAc locus by simplifying an O-sugar chain strategy through an inhibitor and adopting an ultrafiltration-assisted lectin affinity enrichment technology.

Description

High-throughput simple cell O-glycosylation site enrichment and identification method

Technical Field

The invention belongs to the technical field of protein analysis, and particularly relates to a high-throughput simple method for enriching and identifying O-glycosylation sites of cells.

Background

Glycosylation is the most common post-modification in protein modification. The analysis technology of N-glycosylation modification is mature at present, but the analysis of O-glycosylation modification, especially O-GalNAc (O-acetylgalactosamine) modification, still has a great bottleneck. The deletion of highly specific O-sugar chain releasers and the non-conservative modification site sequence characteristics (such as NXS/T motif of N sugar) make the O-GalNAc site research unable to directly follow the existing N-glycosylation technology. Meanwhile, the characteristics of high molecular weight of sugar chains, easiness in breaking glycosidic bonds and the like cause that the library search of O-GalNAc mass spectrum cannot be realized by setting fixed molecular weight modification in the library search process like acetylation, phosphorylation, O-GlcNAc and the like modification. These all limit the large-scale identification of the glycosylation modification sites of O-GalNAc. In recent years, a Simple cell (Simple cell) strategy developed by Clausen et al gene means knocks out chaperone COSMC of O-sugar initiation elongase, simplifies the sugar chain structure of O-GalNAc, simplifies the sugar chain of O-GalNAc produced by cells into the form of only one monosaccharide O-GalNAc, and then identifies the O-GalNAc, thereby obtaining the current largest O-GalNAc site data set. However, the chaperonin gene needs to be stably knocked out, the difficulty of experimental operation technology is high, and the application and popularization of the method are limited to a certain extent. Therefore, the invention adopts a 'chemical simplified cell strategy' to realize large-scale enrichment and identification of O-glycosylation sites, the method specifically inhibits the synthesis of O-GalNAc sugar chains at the level of single GalNAc through an inhibitor, further combines ultrafiltration-assisted lectin affinity enrichment to realize high-flux enrichment of O-GalNAc, and then realizes high-flux and efficient identification of the O-glycosylation sites through the analysis of liquid mass spectrometry.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for enriching and identifying O-glycosylation sites of cells, which is simple, easy to operate, rapid, efficient, high-flux and simple.

The invention provides a high-flux simple method for enriching and identifying O-glycosylation sites of cells, which comprises the following specific steps:

(1) suppressing O-GalNAc sugar chain synthesis at a level of a single GalNAc by adding an inhibitor of O-GalNAc sugar chain synthesis in a cell culture environment;

(2) then the enrichment of O-GalNAc glycopeptide is realized by an ultrafiltration auxiliary lectin enrichment technology, and the enriched O-glycopeptide is released by utilizing a monosaccharide competition mechanism;

(3) and finally, identifying the O-GalNAc glycosylation site through LC-MS in a high-throughput manner.

The invention provides a high-flux simple method for enriching and identifying O-glycosylation sites of cells, which comprises the following further specific operation flows:

(1) adding an inhibitor of O-sugar chain synthesis to a cell culture medium;

(2) culturing the cells in a medium containing an inhibitor;

(3) discarding the culture medium, washing with PBS for 3 times, and performing conventional cell collection, protein lysis and pancreatin enzymolysis;

(4) adding PNGase F and sialidase to remove N-sugar chain and sialic acid;

(5) desalting the enzymatic peptide fragment with C18 small column, and lyophilizing;

(6) incubating the lectin and the lyophilized peptide fragment sample together;

(7) transferring the incubated lectin solution to an ultrafiltration tube, performing ultrafiltration, washing for more than 5 times, and washing off non-glycoprotides which are not combined with the lectin;

(8) adding GalNAc with the final concentration of 0.1-1 mol/L into the system, incubating for 0.5-2 h, and releasing the O-glycopeptide combined with the lectin through monosaccharide competition;

(9) ultrafiltering, washing for 2-5 times, and combining the collected mixed solution of the O-glycopeptide and the GalNAc;

(10) removing salt and GalNAc from the system by desalting with C18 small column, and lyophilizing;

(11) and performing LC-MS analysis to identify the O-GalNAc glycosylation site.

In the present invention, the O-sugar chain synthesis inhibitor used includes, but is not limited to, Benzyl 2-acetamido-2-deoxy-alpha-D galactoside (Benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside).

In the process (1) of the present invention, the medium used is a medium recommended as a source of the cell purchase; the concentration range of the inhibitor added in the step (1) is 1-10 mM.

In the process (2), the cell is cultured in the inhibitor environment for 24-48 h.

In the process (4), the PNGase F and the sialidase are added according to the proportion of 300-500U corresponding to 1 mg of the starting protein, and the PNGase F and the sialidase are incubated at 37 ℃ for 14-18 hours.

In the process (6), the added agglutinin is Viciavilosa exogenous agglutinin (VVL), and the added concentration is 1-5 micrograms/microliter; incubating at room temperature for 0.5-3 h, wherein the pH of the incubated solution and the ultrafiltration solution is 10-100 mM Tris/HCl, the pH of the incubated solution is 7-8, and the pH of the incubated solution and the ultrafiltration solution is 0-2 mM MnCl₂, 0~2mM CaCl₂, 0~2 MNaCl。

The invention adds inhibitor during cell culture, so that the elongation of all O-GalNAc sugar chains in cells is inhibited, all O-GalNAc sugar chains exist in a single GalNAc form, the structure of the sugar chain is simplified, and after sialic acid possibly existing is removed, the O-sugar can be identified by lectin VVL. The enrichment of O-sugar can be realized rapidly and with high flux by means of ultrafiltration auxiliary technology and C18 desalination technology. The invention has the characteristics of simple and rapid operation, high flux and the like.

Drawings

FIG. 1 is a flow chart of the method.

FIG. 2 is a graph showing the results of lectin blotting.

Detailed Description

The process of the present invention is further illustrated by the following specific examples.

Example 1:

VVL and lectin blot corresponding to avidin are incubated by protein extracted before and after adding inhibitor into Hela cells

Adding 2mM, 5 mM and 10mM inhibitors into DMEM medium respectively, incubating with cells for 24h, performing protein extraction after 48h, and performing lectin blotting.

The lectin agglutination procedure was as follows:

(1) electrophoresis: firstly, preparing polyacrylamide gel electrophoresis gel. After the gel was prepared, 20. mu.g of the sample solution was added to the loading buffer, suspended, centrifuged and added to the lane. Additionally marker was added as a molecular weight control. Placing the electrophoresis plate into an electrophoresis tank, and setting current: the current is set to be 10 mA/gel when the concentrated gel is separated, and when the bromophenol blue front reaches the interface of the separated gel, the current is adjusted to be 20 mA/gel. Stopping until the front edge of the bromophenol blue is about 0.5cm away from the bottom of the gel;

(2) the membrane was blocked, incubated with 3. mu.g/mL primary antibody (lectin VVL) and 0.8. mu.g/mL secondary antibody (avidin) for 30 min each before analysis by exposure.

FIG. 2 is a graph showing the results of lectin blotting.

It can be seen that the simplified glycoform is significantly increased after the inhibitor is added, which indicates that the inhibitor can achieve the expected effects of inhibiting the elongation of the sugar chain and generating the single GalNAc glycoform glycoprotein; it can also be seen that the content of the resulting simplified glycoform protein increases with time and with increasing inhibitor concentration. The best effect is achieved by adding 10mM Benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside during the cell culture process and continuing the culture for 48 hours. After inhibition, the O-GalNAc glycoform in the cell can be obviously simplified, and the glycoprotein can be effectively enriched through agglutinin. In addition, the natural O-GalNAc has no fixed composition form, no software can carry out large-scale analysis on mass spectrum data of the natural O-GalNAc, the strategy of simplifying sugar chains exists at present, and conventional mass spectrum analysis software can carry out analysis on O-GalNAc glycoprotein.

Example 2

Large-scale identification of O-GalNAc sites of Hela cells:

10 mMBenzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside inhibitor is added into DMEM medium, cells are washed 3 times by PBS after being incubated for 48h, and then proteolytic cleavage and proteolysis are carried out. PNGase F and sialidase were added at a rate of 500U to 1 mg of starting protein, incubated at 37 ℃ for 16 hours, desalted using a C18 mini-column, and lyophilized. Subsequently, 200. mu.l of VVL and buffer, and of the VVL lectin at a concentration of 2.5. mu.g/l were added to the lyophilized peptide fragments. Incubate at room temperature for 1h, transfer to an ultrafiltration tube, centrifuge at 12000g for 15 min, then add buffer 200. mu.l, centrifuge at 12000g for 15 min, wash 5 times. Then adding GalNAc with the final concentration of 1M, shaking at room temperature for 1.5 h, centrifuging at 12000g for 15 min, washing twice with 100 microliters of buffer solution, combining the solutions obtained by 3 times of ultrafiltration, desalting by using a C18 small column, freeze-drying, and sending to liquid chromatography-mass spectrometry. A total of 215O-GalNAc modified glycoproteins, 224 glycosylated peptide segments and the corresponding 498 sites were identified. The method can quickly, simply and conveniently realize large-scale enrichment and quantification of the O-GalNAc locus, and fills the blank of the existing method.

Claims

1. A method for enriching and identifying high-flux simple cell O-glycosylation sites is characterized by comprising the following specific steps:

(2) then the enrichment of O-GalNAc glycopeptide is realized by an ultrafiltration auxiliary lectin enrichment technology, and the enriched O-GalNAc glycopeptide is released by utilizing a monosaccharide competition mechanism; which comprises the following steps:

(a) adding PNGase F and sialidase to remove N-sugar chain and sialic acid;

(b) desalting the peptide fragment after enzymolysis by using a C18 small column, and then freeze-drying;

(c) incubating the lectin and the peptide segment after desalting;

(d) transferring the incubated lectin solution to an ultrafiltration tube, performing ultrafiltration, washing for more than 5 times, and washing off non-glycoprotide which is not combined with the lectin;

(e) adding GalNAc with the final concentration of 0.1-1 mol/L into the system, incubating for 0.5-2 h, and releasing O-GalNAc glycopeptide combined with the lectin through monosaccharide competition;

the agglutinin is a vicia villosa lectin;

(3) finally, through LC-MS, identifying the O-GalNAc glycosylation site in high flux;

the O-GalNAc sugar chain synthesis inhibitor is Benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside;

the concentration of the inhibitor added in the process (1) is 1-10 mM;

in the process (2), the time for culturing the cells in the inhibitor environment is 24-48 h.

2. The method according to claim 1, wherein the specific operation flow is:

(1) adding an inhibitor of O-GalNAc sugar chain synthesis to a cell culture medium;

(2) culturing the cells in a medium containing an inhibitor;

(4) adding PNGase F and sialidase to remove N-sugar chain and sialic acid;

(5) desalting the peptide fragment after enzymolysis by using a C18 small column, and then freeze-drying;

(6) incubating the lectin and the peptide segment after desalting;

(7) transferring the incubated lectin solution to an ultrafiltration tube, performing ultrafiltration, washing for more than 5 times, and washing off non-glycoprotide which is not combined with the lectin;

(8) adding GalNAc with the final concentration of 0.1-1 mol/L into the system, incubating for 0.5-2 h, and releasing O-GalNAc glycopeptide combined with the lectin through monosaccharide competition;

(9) ultrafiltering, cleaning for 2-5 times, and combining the collected O-GalNAc glycopeptide and GalNAc mixed solution;

(11) and performing LC-MS analysis to identify the O-GalNAc glycosylation site.

3. The method according to claim 2, wherein in the scheme (4), the PNGase F and the sialidase are added in a ratio of 300-500U to 1 mg of the starting protein, and incubated at 37 ℃ for 14-18 hours.

4. The method according to claim 2, wherein in the process (6), the lectin is added at a concentration of 1-5 μ g/μ l; incubating at room temperature for 0.5-3 h, wherein the pH of the incubated solution and the ultrafiltration solution is 10-100 mM Tris/HCl, 7-8, 0-2 mM MnCl 2, 0-2 mM CaCl 2 and 0-2 mM NaCl.