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Magnetic nanometer material, and preparation method and application thereof in enrichment analysis for glycosylation peptide fragment

A magnetic nanometer and magnetic nanoparticle technology, applied in the fields of biotechnology and nanomaterials, achieves the effects of high sensitivity, good selectivity and simple synthesis method

Active Publication Date: 2017-05-31
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to literature research, there is no iron ferric oxide magnetic nanomaterial directly modified by cysteamine hydrochloride used in the enrichment of glycopeptides.

Method used

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  • Magnetic nanometer material, and preparation method and application thereof in enrichment analysis for glycosylation peptide fragment
  • Magnetic nanometer material, and preparation method and application thereof in enrichment analysis for glycosylation peptide fragment
  • Magnetic nanometer material, and preparation method and application thereof in enrichment analysis for glycosylation peptide fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Synthesis of a ferroferric oxide magnetic nanomaterial modified with cysteamine hydrochloride.

[0028] (1) Weigh 2.6g of ferric chloride hexahydrate and disperse it in 145mL of ethylene glycol, stir and mix evenly with magnetic force, add 7.3g of anhydrous sodium acetate to the mixture, continue to stir and mix evenly, and then disperse with ultrasound for 0.5 hours , transfer the mixed solution to a polytetrafluoroethylene-lined stainless steel reaction kettle, react at 200°C for 16 hours, then fully wash with deionized water and absolute ethanol, and dry under vacuum at 50°C to obtain ferric oxide magnetic nanoparticles.

[0029] (2) Weigh 22 mg ferroferric oxide magnetic nanoparticles obtained in step (1), and disperse them into 30 mL of 0.01M phosphate buffer, then add 88 mg of cysteamine hydrochloride, ultrasonically disperse for 5 minutes, and place in a water bath at 60°C After mechanical stirring for 3 hours, the product was magnetically separated f...

Embodiment 2

[0034] Example 2: The ferric iron tetroxide magnetic nanomaterial modified with cysteamine hydrochloride obtained in Example 1 was applied to the enrichment and MALDI-TOF MS detection of low-concentration horseradish peroxidase HRP hydrolyzate.

[0035] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg of HRP standard protein, prepare a standard protein solution with a concentration of 10 mg / mL with 25 mM ammonium bicarbonate solution, boil for 5 minutes; then dilute with 25 mM ammonium bicarbonate solution to make HRP The final concentration is 1mg / mL, according to the mass ratio of 1:40 trypsin and standard protein, add trypsin (trypsin), and incubate at 37°C for 16 hours to obtain 1mg / mL HRP trypsin hydrolyzate.

[0036] (2) Enrichment of samples: Prepare a sample solution with a volume fraction of 85% acetonitrile / 14.9% water / 0.1% trifluoroacetic acid, and use the sample solution to prepare 10 mg / mL cysteamine hydrochloride-modified trioxide Dispersions of f...

Embodiment 3

[0042] Example 3: The ferric iron tetroxide magnetic nanomaterial modified by cysteamine hydrochloride obtained in Example 1 was enriched and eluted for many times, and then applied to the enrichment of low-concentration horseradish peroxidase HRP enzymatic hydrolyzate Set with MALDI-TOF MS detection.

[0043] (1) Prepare standard proteolysis solution: Accurately weigh 1 mg of HRP standard protein, prepare a standard protein solution with a concentration of 10 mg / mL with 25 mM ammonium bicarbonate solution, boil for 5 minutes; then dilute with 25 mM ammonium bicarbonate solution to make HRP The final concentration is 1mg / mL, according to the mass ratio of 1:40 trypsin and standard protein, add trypsin (trypsin), and incubate at 37°C for 16 hours to obtain 1mg / mL HRP trypsin hydrolyzate.

[0044] (2) Enrichment of samples: Prepare a sample solution with a volume fraction of 85% acetonitrile / 14.9% water / 0.1% trifluoroacetic acid, and use the sample solution to prepare 10 mg / mL c...

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Abstract

The invention belongs to the technical field of biotechnology and nanometer material and specifically relates to a ferroferric oxide magnetic nanometer material modified with cysteamine hydrochloride, and a preparation method and an application thereof in enrichment analysis for a glycosylation peptide fragment. According to the invention, the ferroferric oxide magnetic nanometer material modified with cysteamine hydrochloride and a glycosylation peptide fragment solution are mixed in a mixed solution of acetonitrile, water and trifluoroacetic acid; the mixture is hatched in an enzymolysis instrument; the nanometer material is separated and the supernate is removed under an action of an external magnetic field; the nanometer material is washed with the mixed solution of acetonitrile, water and trifluoroacetic acid; the glycosylation peptide fragment enriched on the material is eluted with the mixed solution of acetonitrile, water and formic acid; and the mass spectrometry is combined for identifying. According to the invention, the operation is simple and quick; the material can be reused; the cost is low; the sensitivity and the selectivity are higher; the ferroferric oxide magnetic nanometer material is suitable for the enrichment analysis for the glycosylation peptide fragment and has excellent application prospect.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and nanometer materials, in particular to a magnetic nanometer material and its preparation method and its application in the enrichment and identification of glycosylated peptides. Background technique [0002] Glycosylation of proteins is one of the most important post-translational modifications in the life process. More than 50% of human proteins are glycosylated, which plays an important role in life activities such as cell signal transduction, molecular recognition, and immune response. role. Therefore, a comprehensive study of protein glycosylation is critical for understanding human life processes and disease mechanisms. But the current glycoprotein research still encounters many difficulties. Since the glycosylated proteins are low-abundance proteins with low ionization efficiency, they are easily interfered by non-glycoproteins with high abundance and high ionization efficiency, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/286B01J20/30G01N27/62
CPCB01J20/286G01N27/62
Inventor 张祥民冯小燕
Owner FUDAN UNIV
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