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Method for enriching glycopeptide by phenylboronic acid material

A phenylboronic acid and enrichment technology, applied in the field of separation and purification, can solve problems such as reducing selectivity, and achieve the effects of improving detection limit, wide versatility and high selectivity

Active Publication Date: 2015-02-25
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, hydrogen bonding between nonglycopeptides and boronic acid materials reduces the selectivity of the method

Method used

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  • Method for enriching glycopeptide by phenylboronic acid material
  • Method for enriching glycopeptide by phenylboronic acid material

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Experimental program
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preparation example Construction

[0062] The preparation process of monolayer phenylboronic acid modified silica spheres was L-aspartic acid (4g, 30mmol), K2CO3 (18g, 130mmol), CuSO4·5H2O (0.075g, 0.3mmol), 2-oxazole-1-sulfonyl alkene Nitride (8.5g, 40.6mmol), CH3OH (100mL) and spherical silica gel (20g) with a particle size of 5μm were added to a 250mL round-bottomed flask, and the mixture was stirred at room temperature for 9h, then concentrated and dried by centrifugation to obtain α- alkene Nitrogen L-Aspartate. Then, copper acetate (0.4 g, 2 mmol) and sodium ascorbate (0.8 g, 4 mmol) aqueous solution (100 mL) were added to α-azide L-aspartic acid, and stirred at room temperature for 114 h. The obtained product was successively washed with methanol, water, 10% EDTA aqueous solution, water, and methanol twice, 100ml each time, and vacuum-dried at room temperature for 12 hours to obtain a single-layer phenylboronic acid-modified silicon sphere.

[0063] The preparation process of multilayer phenylboronic ac...

Embodiment 1

[0067] Put 1 mg of phenylboronic acid-modified silicon spheres into the extraction column, equilibrate the column with 30 L of 50 mM ammonium bicarbonate solution containing 80% acetonitrile by volume concentration; digest 10 L of 100 g / mL standard glycoprotein horseradish peroxidase with trypsin The solution was spin-dried, dissolved in 30L of 50mM ammonium bicarbonate solution containing 80% acetonitrile by volume, and loaded onto the extraction column;

[0068] Wash with 30L of 50mM ammonium bicarbonate solution containing 80% acetonitrile to remove non-glycopeptides; repeat the washing process twice;

[0069] Then, 20 L of 50% acetonitrile (pH3) aqueous solution was used to elute the glycopeptide, and the elution process was repeated twice; the enriched glycopeptide fractions were combined and analyzed by mass spectrometry.

[0070] Depend on figure 1 It can be seen that the multilayer phenylboronic acid-modified silicon spheres are specific for the enrichment of glycopep...

Embodiment 2

[0072] Disperse 1 mg of phenylboronic acid-modified silicon spheres with 100 L of 50 mM ammonium bicarbonate solution containing 80% acetonitrile by volume, centrifuge, discard the upper layer solution, and collect the precipitate; 10 L of 100 g / mL standard glycoprotein horseradish peroxidase trypsin After the enzymolysis solution was spin-dried, dissolve it in 100L of 50mM ammonium bicarbonate solution containing 80% acetonitrile by volume, mix it with phenylboronic acid-modified silica gel material, incubate for 0.5-120 minutes (specifically, 30 minutes), centrifuge, and discard the upper layer solution. Collect the precipitate; the centrifuged phenylboronic acid-modified silica gel material is mixed with 100L of 50mM ammonium bicarbonate solution containing 80% acetonitrile and incubated for 0.5-120 minutes, centrifuged, the upper layer solution is discarded, and the precipitate is collected; repeat this step twice;

[0073] After centrifugation, the phenylboronic acid-modif...

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Abstract

The invention discloses a method for enriching glycopeptide by a phenylboronic acid material. The glycopeptide is selectively enriched through optimizing the kind and concentration of an organic solvent, the kind and concentration of a buffer salt, the enrichment temperature and other parameters with phenylboronic acid modified silicon spheres as an enrichment material. The method has the characteristics of high selectivity, high adsorption amount, simple and controllable operation and the like, and is suitable for the selective enrichment of glycopeptides in a biological sample.

Description

technical field [0001] The invention belongs to the technical field of separation and purification, and in particular relates to a method for enriching glycopeptides. technical background [0002] Protein glycosylation is involved in many important regulatory processes in eukaryotic cells, including receptor activation, signal transduction, and cell phagocytosis [Ohtsubo and Marth, Cell, 2006]. The occurrence of many diseases is related to the variation of protein glycosylation, such as malignant tumor [Wang, et al., Glycobiology2006], rheumatoid arthritis [Fournier, et al. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, 2000) chronic Obstructive lung disease [Nihlen, et al., Scandinavian Journal of Clinical & Laboratory Investigation, 2001], and Alzheimer's disease [van Rensburg, et al., Metabolic Brain Disease, 2004]. Therefore, it is very important to characterize the structure of glycoproteins. However, the stoichiometric sites expressed at gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08
Inventor 梁鑫淼李秀玲
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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