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A method for solid-phase enrichment and mass spectrometry analysis of n-sugar chains

A mass spectrometry, sugar chain technology, applied in the field of glycoproteomics and glycomic analysis, can solve the problems of low ionization efficiency, difficult identification by mass spectrometry, lack of hydrophobic groups, etc.

Inactive Publication Date: 2017-10-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on N-glycans faces the following difficulties: First, although there are many types of proteins modified by glycosylation, their abundance is usually low, so the content of N-glycans is relatively low; second, N-glycans Due to the lack of hydrophobic groups and the lack of chargeable groups, the ionization efficiency of the chain in mass spectrometry is often lower than that of proteins or peptides, and it is not easy to be identified by mass spectrometry; third, the microscopic heterogeneity of the N-glycan chain itself leads to The signal dispersion and weakening of sugar chains in mass spectrometry make it more difficult to be identified by mass spectrometry with high sensitivity; fourth, although there are solid-phase enrichment methods such as hydrophilic enrichment columns and graphitized carbon columns, these methods are due to solid-phase carrier The interaction between N-glycans and N-glycan chains is weak, and there is a problem of poor selectivity

Method used

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  • A method for solid-phase enrichment and mass spectrometry analysis of n-sugar chains
  • A method for solid-phase enrichment and mass spectrometry analysis of n-sugar chains
  • A method for solid-phase enrichment and mass spectrometry analysis of n-sugar chains

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Experimental program
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Effect test

Embodiment 1

[0037] N-glycan derivatization experiments

[0038] Prepare 100 μL of 100ng / μL standard sugar chain maltoheptaose (DP7) with methanol, add 0.5-2.5 mg / mL of aminophosphonate 4-aminophenylphosphate sodium salt (AMS), and incubate at 60-90 degrees Celsius for 1-3 hours ; freeze-dry the above sample, and add 50μL-1mL 50% ACN aqueous solution (containing 1-5% TFA) to mix for 0.5-2 hours; separate the magnetic nanomaterials from the solution under the action of an external magnetic field; discard the supernatant Then take 50μL-1mL 50% ACN aqueous solution (containing 1-5% TFA) to wash the material 1-3 times, and collect the solid phase material each time; then use 5-10μL NH 3 ·H 2O solution to re-mix the material; under the action of an external magnetic field, the magnetic nanomaterials were separated from the solution, and 1 μL of the supernatant (the final solution containing N-glycan chains) was spotted on the MALDI target plate, and then dried. Spot an equal volume of DHB mat...

Embodiment 2

[0040] Fe 3 o 4 @Ti 4+ Experiments on the ability to selectively enrich N-glycan chains

[0041] Prepare 100 μL of 10ng / μL standard sugar chain maltoheptaose (DP7) and 1 μg / μL bovine serum albumin (BSA) enzymatic peptide with methanol to obtain a mixture of sugar chains and peptides, add 0.5-2.5mg / mL Aminophosphonate 4-aminophenylphosphate sodium salt (AMS), incubated at 60-90 degrees Celsius for 1-3 hours; freeze-dried the above sample, and added 50μL-1mL50% ACN aqueous solution (containing 1-5% TFA) to mix 0.5-2 hours; separate the magnetic nanomaterials from the solution under the action of an external magnetic field; discard the supernatant, and then take 50μL-1mL 50% ACN aqueous solution (containing 1-5% TFA) to wash the material 1-3 times, Collect the solid phase material each time; re-mix the material with 5-10 μL ammonia solution; separate the magnetic nanomaterials from the solution under the action of an external magnetic field, and take 1 μL of the supernatant (f...

Embodiment 3

[0043] Fe 3 o 4 @Ti 4+ Experiments on the selective ability of N-glycan derivatization and solid-phase enrichment in complex samples of human serum

[0044] Use 10-50mM ammonium bicarbonate aqueous solution to prepare 10μL of 100ng / μL human serum enzymatic peptide mixture, add 1μL of PNGase F enzyme, incubate at 37 degrees Celsius for 12-16 hours, then add 1μL of alkaline phosphatase CIP in Incubate at 37°C for 12-16 hours; lyophilize the above sample, re-dissolve the sample with methanol to a final concentration of 100ng / μL, add 0.5-2.5mg / mL of aminophosphonate 4-aminophenylphosphate sodium salt (AMS), in Incubate at 60-90 degrees Celsius for 1-3 hours; freeze-dry the above samples, and add 50 μL-1mL 50% ACN aqueous solution (containing 1-5% TFA) to mix for 0.5-2 hours; Discard the supernatant, then take 50μL-1mL 50% ACN aqueous solution (containing 1-5% TFA) to wash the material 1-3 times, and collect the solid phase material each time; then re-mix with 5-10μL ammonia sol...

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Abstract

The invention belongs to the field of sugar proteomics and glycomics analysis, and relates to a method for using phosphoro-amidate for deriving an N-carbohydrate chain and using Ti4+modifiaiton magnetic nanoparticles for enriching the derived N-carbohydrate chain. The method comprises the steps that firstly, phosphate radicals are introduced to the N-carbohydrate chain derived from the phosphoro-amidate at the reducing end of the N-carbohydrate chain, then the Ti4+modifiaiton magnetic nanoparticles Fe3O4@Ti4+ are placed into a derived carbohydrate chain solution, the carbohydrate chain is fixed to the magnetic nanoparticles through a chelation reaction between the phosphate radicals in the Ti4+ and the carbohydrate chain, non-carbohydrate chain molecules (such as protein, polypeptide, inorganic salt and the like) not combined with the nanoparticles are cleaned away and removed, and finally the captured carbohydrate chain is disengaged from the materials on the alkaline condition and fed into a mass spectrometry carbohydrate chain. The method is simple in steps, convenient to implement, rapid, efficient and capable of achieving high-sensitive and high-selective mass spectrometry of the N-carbohydrate chain.

Description

technical field [0001] The invention belongs to the field of glycoproteomics and glycomics analysis, and relates to a method for N-sugar chain derivation, solid-phase enrichment and mass spectrometry analysis. The method of the invention can significantly improve the selectivity of N-sugar chain mass spectrometry analysis, and has the characteristics of simple steps, convenient operation, rapidity and the like. Background technique [0002] Protein N-glycosylation is a post-translational modification ubiquitous in organisms. According to research reports, more than 1 / 2 of proteins in organisms will undergo N-glycosylation. The N-sugar chains of glycoproteins have important biological functions, and play an important role in cell recognition and molecular recognition, protein folding, and maintaining the correct conformation of proteins. Studies have also shown that the occurrence and development of many major diseases are often accompanied by changes in the composition and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/08
Inventor 陆豪杰张莹蔡炎宾智超杨芃原
Owner FUDAN UNIV
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