Method for catalyzing isotope labeled N-sugar chain by using endoglycosidase

An endoglycosidase and isotope labeling technology, which is applied in the field of isotope 18O labeling N-sugar chains catalyzed by endoglycosidase, can solve problems such as limited application range, complicated operation, and affecting labeling efficiency, and avoid unfavorable factors, chemical Stable properties and high labeling efficiency

Inactive Publication Date: 2010-12-08
FUDAN UNIV
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Problems solved by technology

However, these methods have certain disadvantages. The first two methods involve chemical reactions and require additional reagents and steps, which not only affect the labeling efficiency, but also produce side reactions, which are very unfavorable for subsequent analysis; Complicated and can only be used for cell samples, limited range of applications

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  • Method for catalyzing isotope labeled N-sugar chain by using endoglycosidase
  • Method for catalyzing isotope labeled N-sugar chain by using endoglycosidase
  • Method for catalyzing isotope labeled N-sugar chain by using endoglycosidase

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Embodiment Construction

[0019] The specific steps of the present invention are further described below in conjunction with examples.

[0020] Endoglycosidases include multiple subtypes, and one of the endoglycosidase subtypes, Endo H, is used as an example below.

[0021] Dissolve denatured glycoprotein samples and endoglycosidase Endo H in 18 In a 50 mmol sodium citrate buffer solution (pH 5.5) prepared with O water, the concentration of glycoprotein in the buffer is 0.1-10 μg / μl, and the concentration of endoglycosidase Endo H in the buffer depends on Depends on the specific enzyme activity.

[0022] The solution is placed in an enzymolysis instrument for enzymolysis, the temperature of the enzymolysis instrument is 37°C, the rotation speed is 800-1200 rpm, and the enzymolysis time is 12-20 hours.

[0023] After the enzymatic hydrolysis is completed, move the solution to a 3k-6k ultrafiltration tube, place the ultrafiltration tube in a centrifuge for ultrafiltration, the temperature of the centri...

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Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for catalyzing an isotope labeled N-sugar chain by using endoglycosidase. Glycoprotein enzymolysis reaction of the endoglycosidase is performed in 18O water, and when the N-sugar chain is digested by enzyme, an isotope 18O molecule is labeled at the reductive tail end of the N-sugar chain. Mass spectrometric detection results prove that the difference between the isotope 18O labeled sugar chain molecular weight and the unlabeled sugar chain molecular weight is 2 Daltons and the isotope 18O molecule is stably labeled at the reductive tail end of the sugar chain. The method is simple and efficient, fills the blank of the sugar chain isotope 18O labeling technology, and can be effectively used for relative quantification of the sugar chain.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, in particular to a method for isotope labeling N-sugar chains, in particular to an isotope catalyzed by endoglycosidase 18 O method for labeling N-glycan chains. Background technique [0002] Protein glycosylation is one of the most prevalent, important, and complex protein post-translational modifications (PTMs) in living organisms. Protein glycosylation modification begins in the endoplasmic reticulum and Golgi apparatus, and after maturation, it may be localized in the plasma membrane and cytoplasm to function; or secreted to the outside of the cell to form secreted proteins. Sugar chains play an important role in biological functions. Sugar chains can affect the structure, location and function of proteins, and affect the recognition, signal transduction and interaction of molecules and cells. In addition, many diseases are also closely related to abnormal glycosylation. In cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00
Inventor 张伟汪泓杨芃原
Owner FUDAN UNIV
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