N-sugar chain relative quantitation method based on 18O mark

A relatively quantitative, sugar chain technology used in analytical chemistry to solve problems such as isotopic peak overlap

Inactive Publication Date: 2012-09-05
FUDAN UNIV
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Problems solved by technology

[0006] The purpose of the present invention is to 18 O-labeled N-glycans provide a relatively quantitative method to resolve isotopic 18 O-labeled sugar chains and unlabeled sugar chains are overlapped by isotope peaks in mass spectrometry

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  • N-sugar chain relative quantitation method based on 18O mark
  • N-sugar chain relative quantitation method based on 18O mark
  • N-sugar chain relative quantitation method based on 18O mark

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Embodiment Construction

[0017] The specific steps of the present invention are further described below in conjunction with examples.

[0018] 100 micrograms of chicken ovalbumin (Ovalbumin) and sucrase (Invertase) were subjected to endoglycosidase hydrolysis labeling ( 18 O water buffer solution) and endoglycosidase enzymolysis without labeling (in ordinary ultrapure water buffer solution), and after the enzymolysis sample was purified by ultrafiltration tube and graphitized carbon column in sequence, it was placed in a freeze dryer dry.

[0019] Redissolve the samples with 10-50 microliters of 50% acetonitrile and 0.1% trifluoroacetic acid in water, and take 1-5 microliters for detection by biological mass spectrometry. Both MALDI source and ESI source can be used, and the resolution of mass spectrometry must be greater than 5000 . Obtain unlabeled sugar chain peak intensity (I a ) and its isotopic peak intensity plus 2 Daltons (I a+2 ), and the labeled sugar chain peak intensity (I b ) and its...

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Abstract

The invention belongs to the technical field of analytical chemistry and particularly relates to an N-sugar chain relative quantitation method based on a 18O mark, concretely comprising the following steps of: mixing an N-sugar chain marked by an isotope 18O produced by catalysis of endoglycosidase with an unmarked N-sugar chain produced by catalysis of the endoglycosidase into a solution according to an equal molar ratio, detecting the peak intensity by biological mass spectrometry, and carrying out relative quantitation after eliminating isotope peak overlapping through calculation. The invention successfully solves the problem of isotope peak overlapping of the N-sugar chain marked by the isotope 18O and the unmarked N-sugar chain in the mass spectrometry, obtains good linearity and lower variation coefficient within a two magnitude order dynamic range, and supplies an efficient revise and calculation method to application of the method for marking the N-sugar chain by the isotope through catalysis of the endoglycosidase in quantitative glycomics.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, in particular to a method for the relative quantification of sugar chains, in particular to a method based on 18 Relative quantification of O-labeled N-glycans. Background technique [0002] Protein glycosylation is one of the most common, important and complex protein post-translational modifications (PTMs) in organisms, which participates in various biological processes and plays important functions in life activities. In medicine, glycoproteins and sugar chains are potential disease molecular markers and drug targets, providing the possibility for early diagnosis and treatment of various diseases. With the in-depth research on glycosylation modification and sugar chains, the qualitative research on glycosylation modifications and sugar chains can no longer meet the needs of biological and medical research, and the specific glycosylation modifications or sugar chains between diffe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/62C12Q1/34
Inventor 汪泓张伟杨芃原
Owner FUDAN UNIV
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