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Hydrophilic chromatographic packing as well as preparation method and application thereof

A chromatographic packing and reaction technology, which is applied in the field of analytical chemistry, can solve problems such as limited affinity, and achieve the effect of increasing the number of identifications, improving affinity and loading capacity, and achieving obvious results

Active Publication Date: 2015-03-18
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used hydrophilic fillers are mostly monolayer zwitterions, amide groups or ethylene glycol structures bonded to the surface of silica gel fillers, so the affinity is relatively limited, which is not conducive to the detection of low-abundance glycopeptides or oligosaccharides in complex samples. Highly selective enrichment and separation

Method used

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  • Hydrophilic chromatographic packing as well as preparation method and application thereof
  • Hydrophilic chromatographic packing as well as preparation method and application thereof
  • Hydrophilic chromatographic packing as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] SI-ATRP method for the preparation of hydrophilic polymer-silica gel hybrid packing flow chart is as follows figure 1 shown.

[0060] 1) Synthesis of 2-glucosaminopropyl methacrylate (GMA-G) monomer: Add 0.2M sulfuric acid to 674.53 μl glycidyl methacrylate (GMA), place it in a water bath at 50°C and heat it for the second step. After the first oxidation reaction for 4 hours, 1.09 g of sodium periodate was added and mixed, and the second oxidation reaction was carried out at room temperature in the dark for 2 hours to obtain oxidized GMA;

[0061] Measure another 20 ml of methanol, gradually add 1.1 g of glucosamine, and stir until dissolved. The obtained oxidized GMA was added dropwise to the continuously stirring methanol solution of glucosamine, stirred at room temperature for schiff base reaction for 4 hours to obtain a yellow transparent solution, and the prepared solution was blown to dryness with nitrogen until it was a paste, and GMA- G monomer, sealed and fil...

Embodiment 2

[0073] Example 2, using the chromatographic filler obtained in Example 1 to enrich glycopeptides

[0074] 1) Take 10 mg of the chromatographic filler prepared in Example 1 and dissolve it in acetonitrile, fill it into a 200ul pipette tip, and precipitate naturally to prepare a solid phase extraction column;

[0075] 2) An appropriate amount of bovine fetuin (Sigma, CAS: 9014-81-7) was weighed and dissolved in 50 mM ammonium bicarbonate solution with a final concentration of 1 μg / μl. Add mercaptoethanol at a final concentration of 10 mM, reduce in a water bath at 56°C for 1 hour, then add IAA and place in the dark for 1 hour to denature the glycoprotein. Take the denatured glycoprotein and add trypsin at a mass ratio of 1:50 (trypsin: protein), place it in a 37°C water bath and incubate for 12 hours, then add 0.1% TFA to inactivate the trypsin to obtain an enzymatic hydrolysis product.

[0076] The obtained enzymatic hydrolysis product was divided into two parts, one part was ...

Embodiment 3

[0081] Embodiment 3, utilize the chromatographic filler obtained in embodiment 1 to carry out the enrichment of oligosaccharide

[0082] 1) Take 10 mg of the chromatographic filler prepared in Example 1 and dissolve it in acetonitrile, fill it into a 200ul pipette tip, and precipitate naturally to prepare a solid phase extraction column;

[0083] 2) Take 50 μg of chicken ovalbumin (Sigma Company, CAS: 9006-59-1) and dissolve it in 50 mM ammonium bicarbonate solution with a final concentration of 1 μg / μl. After thermal denaturation in a boiling water bath for 10 minutes, cool to room temperature, add an appropriate amount of denatured protein to PNGase F (the mass ratio of enzyme and protein is 1:20), and incubate in a 37°C water bath for 16 hours. The enzymatic hydrolyzate was lyophilized and dissolved in acetonitrile / water / formic acid (80:20:0.1, v / v) with a final concentration of 1 μg / μl. Rinse the solid-phase extraction column with 30 μl acetonitrile / water / formic acid (10:...

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Abstract

The invention discloses a hydrophilic chromatographic packing as well as a preparation method and application thereof. The packing is hydrophilic polymer-silica gel hybridization packing generated by in-situ polymerizing GMA-G hydrophilic monomers on the surface of silica gel particles through a surface-initiated atom transfer radical polymerization method. The surface of the hybridization packing is highly coarse, so that the specific surface area is relatively high. The concentration and separation of glycopeptides or oligosaccharide in a standard glycoprotein, oligosaccharide and plasma complicated sample can be successfully realized by utilizing a solid phase extraction column and a high efficiency liquid phase chromatographic column, which are filled with the hydrophilic polymer-silica gel hybridization packing. The selective concentration and separation effect is obvious, and 47 varieties of oligosaccharide glycoforms can be identified in the plasma glycoprotein; moreover, the desalting treatment on the sample can be realized in the concentration process, and the loss of the sample can be reduced. The application value in the separation analysis of the complicated biological sample is relatively good.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and relates to a hydrophilic chromatographic filler and a preparation method and application thereof. Background technique [0002] Glycosylation modification of protein is one of the most common and important post-translational modifications, and more than 50% of proteins in the human body are glycosylation modified proteins (Jensen, O.N.Nat.Rev.Mol.Cell Biol.2006,7,391-403 .). The realization of many protein functions, such as immune response, cell recognition, adhesion and migration, etc. are closely related to glycosylation modification (Bertozzi, C.R. et al. Science2001, 291, 2357-2364. Ohtsubo, K. et al. Cell2006, 126, 855-867.). Glycosylated proteins also play an important role in the diagnosis of diseases. Currently known clinical diagnostic markers and therapeutic targets for many diseases, such as CD44 in breast cancer, prostate specific antigen in prostate cancer, ovarian cancer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/26B01J20/281B01J20/30B01D15/08G01N30/02C08F292/00C08F220/36G01N30/08G01N30/14
CPCB01D15/08B01J20/262B01J20/281B01J20/3085B01J2220/46B01J2220/4806B01J2220/4812B01J2220/80C08F220/36C08F220/365C08F292/00G01N30/08
Inventor 钱小红秦伟捷潘一廷
Owner ACADEMY OF MILITARY MEDICAL SCI
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