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Magnetic nano material for enriching nitrine-mark bio-macromolecules and preparation and application of magnetic nano material

A magnetic nano and magnetic technology, which is applied in the preparation method of peptides, the magnetism of organic materials/organic magnetic materials, nanotechnology, etc., can solve the problems of inconvenient operation and many operation steps.

Active Publication Date: 2012-04-11
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these enrichment methods have the disadvantages of many steps and inconvenient operation.

Method used

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  • Magnetic nano material for enriching nitrine-mark bio-macromolecules and preparation and application of magnetic nano material
  • Magnetic nano material for enriching nitrine-mark bio-macromolecules and preparation and application of magnetic nano material
  • Magnetic nano material for enriching nitrine-mark bio-macromolecules and preparation and application of magnetic nano material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Embodiment 1, the preparation of the magnetic nano material that is used for enrichment:

[0140] See figure 1 , as detailed below:

[0141] 1) Superparamagnetic Fe 3 o 4 Preparation of magnetic bead cores: Add 3.0 g FeCl to 80 mL ethylene glycol 3 ·6H 2 O, stirred at room temperature for 30 min, then added 4.8 g of anhydrous sodium acetate and 0.72 g of trisodium citrate, and continued stirring for 1 h. Pour the mixed solution into a 100mL polytetrafluoroethylene reaction cup and seal it in a stainless steel reaction kettle, and heat at 200°C for 8-12h. After cooling to room temperature, the magnetic beads were precipitated under magnetic force, and the finally obtained Fe was washed 3 times with ethanol and water. 3 o 4 magnetic beads.

[0142] 2) Preparation of carboxyl magnetic beads: 10-700 mg of Fe prepared in step 1) 3 o 4 Put the magnetic beads in a 50mL centrifuge tube, add 3mL concentrated ammonia water (25-28wt%), 50mL deionized water and 150mL 95% ...

Embodiment 2

[0145] Embodiment 2, preparation of standard GalNAz glycosylated polypeptide

[0146] The standard GalNAz glycosylated polypeptide is prepared by azide N- Acetylgalactosaminoside (GalNAz) glycosylation modification reaction is completed. The reaction was carried out in the presence of 25mM Tris-HCl (pH 7.4), 5mM MnCl 2 and a 10 μL reaction system of 0.2% (v / v) Triton X-100, the sugar donor substrate of this reaction system is UDP-GalNAz (250mM, synthesized by Shanghai Institute of Organic Chemistry), and the acceptor substrate is Muc5Ac- FAM (25mM), ppGalNAc-T2 was 25ng in each reaction system. After reacting at 37°C for 12 hours, the reaction was terminated by heating at 95°C for 3 minutes.

[0147] After the reaction, the purified GalNAz glycosylated Muc5ac-FAM glycopeptide was separated by HPLC. The specific operation is as follows: dilute 10 μL of the reaction solution to 50 μL, load 20 μL of the sample, and separate through a Cosmosil 5C18-AR reversed-phase analytical...

Embodiment 3

[0167] Example 3, Capture, Release and Mass Spectrometry Detection of GalNAz Modified Polypeptides

[0168] 1. Capture and release of GalNAz modified peptides

[0169] Standard GalNAz glycopeptides with different concentrations (0, 5, 10, 20 pmol) were dissolved in 200 μL of 1% Triton X-100 and 10 mM phosphate buffered saline (PBS) with a pH value of 7.8, and mixed with 100 μg of KSM magnetic beads Mix well. Sodium ascorbate, copper sulfate, and TBTA were added so that the final concentrations of the standard GalNAz glycopeptides were 2 mM, 1 mM, and 0.1 mM, respectively. React at 25°C and 1400rpm shaking for 3 to 24 hours ( figure 2 ).

[0170] TBTA: Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, the structural formula is as follows:

[0171]

[0172] Under the action of magnetic force, collect the magnetic beads, remove the supernatant, wash twice with washing solution (containing 1% Tween (v / v), 0.2wt% SDS, 0.5M NaCl and 25mM Tris7.4, solvent is water), Wash wi...

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Abstract

The invention provides a novel surface chemical modification magnetic material and a method for enriching nitrine-mark bio-macromolecules by using the novel surface chemical modification magnetic material. In the invention, a magnetic nano material is combined with an isolation strategy in the prior art, and a terminal alkynyl is fixed on a super-paramagnetism magnetic bead through a breakable radical to form a novel surface modification magnetic bead containing the terminal alkynyl. In addition, through a Cu(I) catalyzed click chemical reaction, a nitrine radical and the alkynyl on the surface of the magnetic bead form a covalent bond; a covalently bonded material is enriched and separated by using super-paramagnetism of the magnetic bead; and finally, the bio-macromolecules containing nitrine marks are dissociated by breaking the breakable radical on the surface of the magnetic bead so as to fulfill the aim of enriching the nitrine radical marked bio-macromolecules. Compared with the prior art, the advantages of simplified operation and increased efficiency are provided.

Description

technical field [0001] The present invention relates to a magnetic nano material enriched with azide labeled biological macromolecules and its preparation and application, in particular to a magnetic nano material based on alkynyl and disulfide bond modification and the use of the material to enrich azide A new method for nitrogen labeling of biomacromolecules. Background technique [0002] 1. Protein O-GalNAc glycosylation and bioorthogonal metabolic labeling in vivo [0003] Glycosylation of proteins is a modification in which sugar chains (or single monosaccharides) are bound to specific amino acid residues in proteins under the action of glycosyltransferases. It is estimated that more than 50% of proteins in cells have glycosylation [Hagglund, P., et al., A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. Journal of Proteome Research, 2004.3(3): p.556-5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): H01F1/42B82Y25/00C07K1/14
Inventor 张延王胜谢文娴
Owner SHANGHAI JIAO TONG UNIV
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