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Immobilized metal ion affinity chromatography microsphere material as well as preparation and application thereof

A technology for immobilizing metals and microspheres is applied in the field of immobilized metal ion affinity chromatography materials, which can solve the problems of unfavorable large-scale production and practical application, harsh reaction conditions, and complicated preparation process.

Active Publication Date: 2021-09-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its complex preparation process and harsh reaction conditions are not conducive to large-scale production and practical application

Method used

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  • Immobilized metal ion affinity chromatography microsphere material as well as preparation and application thereof
  • Immobilized metal ion affinity chromatography microsphere material as well as preparation and application thereof
  • Immobilized metal ion affinity chromatography microsphere material as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Ti with porous polymer microspheres as matrix 4+ -IMAC material for the separation and enrichment of phosphorylated peptides in biological samples

[0028] Ti based porous polymer microspheres 4+ -Preparation of IMAC microsphere material:

[0029] Add 60mg of PVA into a mixture of 50mL of water and 10mL of toluene, heat to 60°C to dissolve it completely, cool the solution to room temperature, then add 4.7mL of allyl glycidyl ether and 2.9mL of pentaerythritol tetraacrylate dropwise, and finally add 80mg AIBN was used as initiator. Stir mechanically at a rate of 450 rpm in a 70°C oil bath for 2h and then rise to 90°C for 30min to obtain shaped polymer microspheres. After the reaction, the product was washed three times with ethanol and water respectively, the residual solvent was removed by suction filtration, and vacuum-dried at 50° C. for 6 hours to obtain epoxy polymer microspheres.

[0030]Weigh 0.5g of O-phospho-L-serine in a centrifuge tube, add 18mL ...

Embodiment 2

[0042] Example 2 Commercial IMAC Microspheres for the Separation and Enrichment of Phosphorylated Peptides

[0043] Commercial IMAC microsphere material chelates titanium:

[0044] Take 0.5 g of commercial IMAC microspheres (purchased from Sigma-Aldrich), carry out chelation with 5 g of titanium sulfate in aqueous solution at a ratio of 1:10 for 16 h, and then use 10 mL of volume concentration 0.1% TFA, 200 mM NaCl aqueous solution and deionized water The remaining titanium ions were washed away, and vacuum-dried at 50°C for 6 hours to obtain a commercial Ti-IMAC material.

[0045] Commercial Ti-IMAC material enriches phosphorylated peptides:

[0046] The enrichment of phosphorylated peptides was carried out with commercial Ti-IMAC, and the specific operation and conditions of the experiment were the same as in "Example 1".

[0047] Such as Figure 5 As shown, the two materials were used simultaneously for the enrichment of phosphopeptides in the β-casein hydrolyzate. Befo...

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Abstract

The invention relates to an immobilized metal ion affinity chromatography material taking epoxy polymer microspheres as a matrix and application of the immobilized metal ion affinity chromatography material in enrichment of phosphorylated peptides. Specifically, firstly, allyl glycidyl ether and pentaerythritol tetraacrylate are used as raw materials, porous polymer microspheres with epoxy groups are prepared through a suspension polymerization method; the porous polymer microspheres are used as a matrix, O-phosphate-L-serine with phosphate radical functional groups is used as a modifier, a phosphoric acid functional group is introduced to the surface of the material through an epoxy-ammonia ring-opening reaction under an alkaline condition, and finally the Ti < 4 + >-IMAC material capable of being used for enriching phosphopeptides is obtained through chelating titanium ions. The obtained Ti < 4 + >-IMAC material has very high selectivity on the phosphopeptides, and can be used for high-sensitivity and high-selectivity enrichment of endogenous phosphopeptides in biological sample beta-casein enzymatic hydrolysate and human serum.

Description

technical field [0001] The invention relates to an immobilized metal ion affinity chromatography (Ti 4+ -IMAC) materials and their applications in proteomics. The specific preparation process is: [0002] First, porous epoxy microspheres were prepared by suspension polymerization, and then O-phospho-L-serine monomer was introduced into the surface of microspheres by epoxy-amine ring-opening reaction, and then combined with Ti 4+ Chelate, and finally get Ti 4 +-IMAC microsphere material. Background technique [0003] Phosphorylation modification is an extremely important type of post-transcriptional modification in proteomics, and plays an extremely important role in various cellular physiological activities and processes (Document 1, Ptacek, J.et.al "Global analysis of protein phosphorylation in yeast" , Nature, 2005, 438, 679-684.). Mass spectrometry-based proteomics methods are one of the most commonly used and effective tools for studying phosphorylated peptides. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/286B01J20/30C07K1/22G01N27/64G01N30/08
CPCB01J20/286C07K1/22G01N33/6848G01N30/08
Inventor 马淑娟唐睿智欧俊杰叶明亮
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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