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A Glycopeptide Enrichment Method Based on Amphiphilic Porous Core-Shell Microspheres

A porous core and amphiphilic technology, applied in the field of medical testing, can solve the problems of low efficiency and achieve the effect of simple process and easy control of conditions

Active Publication Date: 2020-09-18
TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, only relying on hydrophilic materials to enrich glycopeptides still has the problem of low efficiency

Method used

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  • A Glycopeptide Enrichment Method Based on Amphiphilic Porous Core-Shell Microspheres
  • A Glycopeptide Enrichment Method Based on Amphiphilic Porous Core-Shell Microspheres

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres

[0034] A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres, comprising the following steps:

[0035] 1) In the dSPE mode, put 50 μL of macroporous polydivinylbenzene-polyisopropylacrylamide core-shell microspheres (particle size 6 μm, average pore size 35 nm) into EP tubes, and wash them with 150 μL 20% acetonitrile / formic acid - Ammonium formate buffer solution (buffer salt concentration 5 mM, pH=4.8) and formic acid-ammonium formate buffer solution (buffer salt concentration 5 mM, pH=4.8) activation and equilibration. Dissolve the sample (10 μL desalted and spin-dried fetuin hydrolyzate (1 mg / mL) and 300 μg bovine serum albumin) in 1 mL formic acid-ammonium formate buffer solution (buffer salt concentration is 5 mM, pH=4.8), and equilibrate The final macroporous polydivinylbenzene-polyisopropylacrylamide core-shell microspheres wer...

Embodiment 2

[0037] Example 2 A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres comprises the following steps:

[0038] 1) In the dSPE mode, put 30 μL macroporous polydivinylbenzene-polyisopropylacrylamide core-shell microspheres (particle size 10 μm, average pore size 20 nm) into EP tubes, and wash them with 150 μL 40% acetonitrile / acetic acid - Ammonium acetate buffer solution (20 mM buffer salt concentration, pH=6.0) and acetic acid-ammonium acetate buffer solution (20 mM buffer salt concentration, pH=6.0) activation and equilibration. Dissolve the sample (10 μL desalted spin-dried fetuin hydrolyzate (1 mg / mL) and 300 μg bovine serum albumin) in 1 mL acetic acid-ammonium acetate buffer solution (buffer salt concentration is 5 mM, pH=6.0), and equilibrate The final macroporous polydivinylbenzene-polyisopropylacrylamide core-shell microspheres were mixed evenly, ...

Embodiment 3

[0040] Example 3 A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres

[0041] A method for enriching glycopeptides based on amphiphilic porous core-shell microspheres, comprising the following steps:

[0042] 1) In the dSPE mode, put 20 μL of macroporous polydivinylbenzene-polymethacrylamide core-shell microspheres (particle size 100 μm, average pore size 500 nm) into EP tubes, buffer with 2000 μL 50% acetonitrile ammonium carbonate solution (buffer salt concentration 20 mM, pH=8.1) and 3% acetonitrile / ammonium carbonate buffer solution (buffer salt concentration 20 mM, pH=8.1) to activate and equilibrate the material. Dissolve the sample (10 μL desalted and spin-dried fetuin hydrolyzate (1 mg / mL) and 300 μg bovine serum albumin) in 1.5 mL 3% acetonitrile / formic acid-ammonium formate buffer solution (buffer salt concentration is 5 mM, pH=4.8) Mix well with the equilibrated macroporous polydivinylbenzene-polyisopropylacrylamide core-shell m...

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Abstract

The invention discloses a glycopeptide enrichment method based on amphipathic porous nuclear shell microspheres. The method includes the steps of firstly, conducting activating with an eluant 1 and balancing with a sample loading solution 1 on large-pore nuclear shell microspheres in sequence by means of a dispersed solid-phase extraction mode, dissolving a sample in the sample loading solution 1,mixing the solution with the balanced large-pore nuclear shell microspheres, conducting incubating and centrifuging to obtain supernate 1, and spin-drying the supernate 1 to obtain powder 1; secondly, conducting flushing with an eluant 2 and balancing with a sample loading solution 2 on the large-pore nuclear shell microspheres in sequence by means of a dispersed solid-phase extraction mode, dissolving the powder obtained through spin-drying in the first step in the sample loading solution 2, mixing the solution with the balanced small-pore nuclear shell microspheres, conducting incubating and centrifuging, abandoning supernate to obtain precipitates 2, flushing the precipitates 2 with leacheate 2, conducting centrifuging again after oscillating, abandoning supernate to obtain small-porenuclear shell microspheres adsorbing glycopeptides, eluting the glycopeptides adsorbed on the small-pore nuclear shell microspheres with the eluant 2, and concentrating the glycopeptides. The method is widely suitable for concentrating a trace amount of glycopeptides in a large amount of protein and non-glycopeptide samples.

Description

technical field [0001] The invention relates to medical detection technology. More specifically, it relates to a glycopeptide enrichment method based on amphiphilic porous core-shell microspheres. Background technique [0002] The occurrence of many major human diseases is often associated with the overexpression of endogenous glycopeptides. Such as depression, cancer, Alzheimer's disease, etc. Therefore, the detection of endogenous glycopeptides is of great significance. However, the content of endogenous glycopeptides in body fluids is very small (the abundance is on the order of pg / mL), and its detection is greatly affected by high-abundance proteins (proteins in blood are about 30-50 mg / mL) and non-glycopeptides. Before analyzing and detecting glycopeptides, the glycopeptides should be separated and enriched from the mixture of proteins and non-glycopeptides. At present, the methods for glycopeptide enrichment mainly include: ultrafiltration, organic solvent precipit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22
CPCC07K1/22
Inventor 王树涛宋永杨樊俊兵梁鑫淼李秀玲
Owner TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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