Material and method for enriching glycosylated protein and glycopeptide in urine

A glycoprotein enrichment technology, applied in the field of protein or peptide enrichment, can solve the problems of inhomogeneity, limited abundance, and low glycosylation modification ratio, and achieve high selectivity, improved hydrophilic ability, and improved binding ability. Effect

Pending Publication Date: 2022-04-08
武汉承启医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the abundance of protein glycosylation itself is limited, and the ratio of glycosylation modification in urine is low and highly micro-heterogeneous, which is easily interfered by high-abundance non-glycosylated proteins and peptides in mass spectrometry analysis. Therefore, To achieve high-sensitivity mass spectrometry identification of glycoproteins / glycopeptides in urine, it must be enriched with high efficiency and high selectivity

Method used

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  • Material and method for enriching glycosylated protein and glycopeptide in urine
  • Material and method for enriching glycosylated protein and glycopeptide in urine
  • Material and method for enriching glycosylated protein and glycopeptide in urine

Examples

Experimental program
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Effect test

Embodiment 1

[0057] This example describes the metal-organic framework Fe 3 o 4 The preparation method of / chitosan.

[0058] Weigh 2mgFe 3 o 4 Pour the powder into a test tube with 4ml of citric acid solution left, ultrasonically shake for 5h, then vortex for 1min, and use a pipette to absorb 1ml of Fe 3 o 4 The suspension was added to a test tube containing 4ml of double distilled water, and ultrasonically oscillated for 1h to obtain Fe 3 o 4 Nanoparticle sol. Pipette 250 μl nonylphenol ethoxylate into 1ml Fe 3 o 4 In the nanoparticle sol, shake slightly to mix the two fully, then heat the mixture into 1ml of chitosan solution with a mass concentration of 0.8mg / ml, vortex for 4s and then stand at room temperature. The final product is clear and transparent Fe 3 o 4 / chitosan composite nanoparticle sol.

[0059] Among them, nonylphenol polyoxyethylene ether can also be replaced by alkyl polyglycoside, Span or Tween.

Embodiment 2

[0061] This example describes the metal-organic framework Fe 3 o 4 The preparation method of / chitosan.

[0062] Measure 9ml of absolute ethanol and 1ml of formic acid in a beaker, put the beaker with solvent in ice water to cool; weigh 0.9g of polyvinylpyrrolidone, 0.1g of chitosan powder and slowly add it to the solvent under stirring, Continue to stir for 1 hour until it swells completely; place the beaker in a water bath, slowly heat it to 80°C under reflux condensation, shake for 24 hours until it dissolves completely, and the polymer solution is transparent, which is used as the shell spinning solution.

[0063] Measure 10ml of absolute ethanol and put it in a beaker, put the beaker with solvent in ice water to cool; weigh 1gPVP / Fe 3 o 4 (PVP and Fe 3 o 4 The mass ratio is 9:1) dissolved in absolute ethanol, slowly added to the solvent under stirring, and vigorously mechanically stirred for 1 hour until it swelled completely, as the core spinning solution.

[0064] ...

Embodiment 3

[0066] This example describes the preparation method of glycoprotein / glycopeptide enrichment material in urine.

[0067] 5 g magnetic metal organic framework Fe 3 o 4 / Suspend chitosan in 30mL isopropanol and stir evenly, add 20mL terephthalic acid solution dropwise under mechanical stirring, continue the self-assembly reaction at room temperature for 3 hours, pour the reaction solution into a centrifuge tube, and centrifuge under the condition of an external magnetic field , washed alternately with N,N-dimethylformamide and absolute ethanol three times, and vacuum-dried to obtain the target product.

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Abstract

The invention relates to the technical field of protein or polypeptide enrichment, and discloses a material and a method for enriching glycosylated protein and glycopeptide in urine. The enrichment material is composed of a metal organic framework and a ligand loaded on the metal organic framework, the metal organic framework is Fe3O4 / chitosan magnetic composite nanoparticles, and the ligand contains at least one carboxyl group. Under the action of an external magnetic field, the enrichment material is used for carrying out centrifugal enrichment on an enzyme digestion product of urine protein, due to the fact that the enrichment material has a plurality of hydrophilic groups, separation and enrichment of hydrophilic glycoprotein / glycopeptide can be achieved based on the similar compatibility principle, and the enrichment material has a magnetic inner core, so that the separation and enrichment of the hydrophilic glycoprotein / glycopeptide can be achieved. And the centrifugal time can be shortened through an external magnetic field, and higher selectivity, sensitivity and enrichment efficiency are achieved.

Description

technical field [0001] This application relates to the technical field of protein or polypeptide enrichment, in particular to a material and method for enriching glycosylated proteins and glycopeptides in urine. Background technique [0002] Glycosylation modification of protein is the most common and important post-translational modification, which plays an important role in biological processes such as protein folding, signal transduction, molecular recognition and immune response. Under the catalysis of enzymes, different glycans are produced. These sugars are mainly distributed on the cell surface and in the extracellular matrix. The study of proteomics helps to explore the mechanism of cancer, which is of great significance to the early detection and prevention of cancer, especially in the invasion and metastasis of tumor cells. Therefore, glycosylated protein, as an important biomarker candidate, has received more and more attention from the scientific and medical com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30B01J20/28C12P21/06C07K1/14
Inventor 郭可夫刘涛
Owner 武汉承启医学检验实验室有限公司
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