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High-resolution sequential separation and accurate quantitative analysis of sialylated sugar chain isomers

A technique of isomerization and sialylation, applied in the field of glycobiology, can solve problems such as difficult quantitative analysis and complex mass spectra, and achieve the effects of improving sensitivity, eliminating influence, and achieving high-resolution separation

Active Publication Date: 2021-04-20
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method greatly simplifies the identification of α2-6 and α2-3 linked sialic acids, but when quantitatively analyzing samples containing multiple sialic acid sugar chains through isotope labeling, the requirements for mass spectrometry resolution will be high, and The mass spectrum will be highly complex, which will cause great difficulties for subsequent quantitative analysis
[0005] To sum up, the main problem of the existing sialylated sugar chain research technology is that it is impossible to achieve accurate quantitative analysis at the level of sialylated sugar chain isomers

Method used

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  • High-resolution sequential separation and accurate quantitative analysis of sialylated sugar chain isomers
  • High-resolution sequential separation and accurate quantitative analysis of sialylated sugar chain isomers
  • High-resolution sequential separation and accurate quantitative analysis of sialylated sugar chain isomers

Examples

Experimental program
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Effect test

Embodiment 1

[0060]A standard glycoprotein IgG sialized n-sugar chain ligation isomer, studies the sialized N-sugar chain of the cow IgG standard sugar protein, the specific steps are as follows:

[0061]S1, scaled with two parts of 5 mg of cow IgG standard sugar protein, soluble in 500 μl of protein-denatured liquid, 100 ° C for 10 min, and after the sample was cooled, 50 μl of phosphate buffer (pH 7.5), 50 μl 10% NP-40, respectively, 50 μl 10% NP-40, respectively. (v / V) solution and 2 μl of pngase f enzyme, at 37 ° C for 24 h. The released N-sugar chain was purified by the C18 solid phase extraction column and graphite carbon solid phase to give a purified N-sugar chain sample;

[0062]Among them, the protein-denatched liquid is 0.4 m of dithioseol (DTT) and 5% dodecyl sodium sodium (SDS); phosphate buffer is 1 M sodium phosphate, pH value of phosphoric acid to 7.5; 2 μL PNGase F Enzyme is 1000 enzyme unit.

[0063]The purification process of the C18 solid phase extract is: C18 solid phase extraction...

Embodiment 2

[0081]A qualitative method of standard sugar protein sialicization N-sugar chain ligation isomers, studies the sialized n-sugar chain of rabbit IgG standard sugar proteins, and the specific steps are as follows:

[0082]S1, scaled with two parts of 5 mg rabbit IgG standard sugar protein, soluble in 500 μl of protein-denaturing liquid, 100 min, 100 ° C, 10 min, 50 μl of phosphate buffer (pH 7.5), 50 μl of 10% NP-, respectively, after cooling 40 (v / v) solution and 2 μl of pngase f enzymes were reacted at 37 ° C for 24 h. The released N-sugar chain was purified by the C18 solid phase extraction column and graphite carbon solid phase to give a purified N-sugar chain sample;

[0083]Among them, the proteolytic liquid is 0.4 m of sodium sugar alcohol (DTT) and 5% dodecyl sulfonate (SDS); phosphate buffer is 1 M sodium phosphate, and the phosphoric acid pH is 7.5; 2 μL PNGase F Enzyme is 1000 enzyme unit.

[0084]The purification process of the C18 solid phase extract is: C18 solid phase extracti...

Embodiment 3

[0102]Quantitative method of standard sugar protein siallation N-sugar chain homide, the study of standard sugar protein IgG and rabbit Ig sialized N-sugar chain, the specific steps are as follows:

[0103]S1, respectively, 9 parts of 5 mg of standard sugar protein IgG and rabbit IgG, soluble in 500 μl of protein-denatured liquid, 10 min, 100 ° C, after the sample is cooled, 5 μl of phosphate buffer (pH 7.5), 50 μl 10%, respectively NP-40 (V / V) solution and 2 μl of PNGase Fase, at 37 ° C for 24 h. The released N-sugar chain was purified by the C18 solid phase extraction column and graphite carbon solid phase to give a purified N-sugar chain sample;

[0104]Among them, the protein-denatched liquid is 0.4 m of dithioseol (DTT) and 5% dodecyl sodium sodium (SDS); phosphate buffer is 1 M sodium phosphate, pH value of phosphoric acid to 7.5; 2 μL PNGase F Enzyme is 1000 enzyme unit.

[0105]The purification process of the C18 solid phase extract is: C18 solid phase extraction column is activate...

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Abstract

The present invention proposes a method for high-resolution sequential separation and accurate quantitative analysis of sialylated sugar chain isomers, comprising: using PNGase F enzyme to release N-sugar chains on glycoproteins; Amidation method is used to label isotope reagent (d0 / d5‑) aniline to protect and stabilize isotope labeling of sialic acid; use solid phase extraction technology (C18 column) to remove neutral sugar chains to avoid the interference of neutral sugar chains; saliva The reducing end of the acidified sugar chain is then labeled with Girard reagent P(GP) to shield the active aldehyde group and improve the detection sensitivity of mass spectrometry; the specific derivatization technology of sialic acid is used to identify the connection isomer; finally, RP‑HPLC‑MS The technology distinguishes, identifies and quantitatively analyzes the isomers of the above-mentioned processed sialylated sugar chain derivatives. This method has strong versatility and for the first time realized the accurate quantitative analysis of sialylated sugar chains at the level of isomers.

Description

Technical field[0001]The present invention belongs to the field of sugar biological technology, and more particularly to a method of relatively quantify the sialicized sugar chain at isomer levels.Background technique[0002]The sugar chain plays an important role in cell recognition, cytosol, bacterial infection, signal transduction, immune response, etc. in the form of free oligosaccharides, glycolid and glycoproteins, and has a variety of disease biological marks. The potential of things, so the relevant research of sugar chains is being received more and more attention. Sialic acid, a group of 9 carbose structural units, located in many free-free o-sugar chains, N-sugar chains, O-sugar chains, and glycoprochemical non-reduction ends, for the stability of the sugar chain structure and biofilter Significance. There are generally two sialic acid forms in mammals, N-acetyl neurine (NEU5ac) and N-hydroxyacetamine (NEU5GC), but only N-acetyl neurine (neu5ac) in the normal portion of the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/89G01N30/06
CPCG01N30/06G01N30/89G01N2030/062
Inventor 王仲孚王承健晋万军黄琳娟
Owner NORTHWEST UNIV
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