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Low-cost, simple and rapid glycoprotein N-carbohydrate chain analysis method

A low-cost technology for glycoproteins, applied in the field of biochemistry, can solve the problems of weak N-glycan mass spectrometry signals and few types of sugar chains, and achieve the effects of increasing analytical abundance, simple operation, and stable and reliable analysis results

Pending Publication Date: 2019-04-16
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mass spectrum signal of the final N-glycan chain is weak, and the number of sugar chain types identified is relatively small

Method used

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  • Low-cost, simple and rapid glycoprotein N-carbohydrate chain analysis method
  • Low-cost, simple and rapid glycoprotein N-carbohydrate chain analysis method
  • Low-cost, simple and rapid glycoprotein N-carbohydrate chain analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 N-glycan analysis of glycoprotein immunoglobulin A1 (IgA1) in human blood

[0038] 1. Method

[0039] (1) 100 μg of IgA1 obtained by separation and purification from human blood was dissolved in 200 μL of 50 mmol / L ammonium bicarbonate (NH 4 HCO 3 ) solution, and denatured by heating at 100°C for 10 min.

[0040] (2) Dithiothreitol (DTT) at a final concentration of 20 mmol / L was added, shaken at 56°C for 45 min, and iodoacetamide (IAM) at a final concentration of 50 mmol / L was added and reacted at room temperature for 1 h in the dark.

[0041] (3) All the reaction solution was added to a 30KD ultrafiltration tube, centrifuged at 13000g for 15min×3 times, each time with 200μL of 50mmol / L NH 4 HCO 3 rinse.

[0042] (4) Add 100 μL of 50 mmol / L NH 4 HCO 3 and 5U (1:20) PNGase F protease solution, and continue to react in a shaker at 37°C for 2h.

[0043] (5) Centrifuge at 13,000 g for 15 min x 3 times to collect N-glycan chains, and wash each time with 200...

Embodiment 2

[0055] Example 2 N-glycan analysis of glycoprotein uromodulin (UMOD) in human urine

[0056] 1. Method

[0057] (1) Dissolve 100 μg of UMOD obtained by separating and purifying human urine into 200 μL of 50 mmol / L ammonium bicarbonate (NH4HCO3) solution, and heat at 100° C. for 10 minutes for denaturation.

[0058] (2) Dithiothreitol (DTT) at a final concentration of 20 mmol / L was added, shaken at 56°C for 45 min, and iodoacetamide (IAM) at a final concentration of 50 mmol / L was added and reacted at room temperature for 1 h in the dark.

[0059] (3) All the reaction solution was added to a 30KD ultrafiltration tube, centrifuged at 13000 g for 15 min x 3 times, and washed with 200 μL of 50 mmol / L NH4HCO3 each time.

[0060] (4) Add 100 μL of 50 mmol / L NH4HCO3 and 5U (1:20) PNGaseF protease solution, and continue to react for 2 h in a shaker at 37°C.

[0061] (5) Centrifuge at 13,000 g for 15 min x 3 times to collect N-glycan chains, and wash each time with 200 μL of ultrapure...

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Abstract

The invention discloses a low-cost, simple and rapid glycoprotein N-carbohydrate chain quantitative analysis method. The method comprises the following steps of (1) carrying out reduction alkylation treatment after the heat denaturation of glycoprotein; (2) using a PNGase F protease to excise an N-carbohydrate chain on glycopeptide in an ultrafiltration tube, and collecting the N-carbohydrate chain in a centrifugation mode; (3) using hydrophilic interaction chromatography to purify and enrich the N-carbohydrate chain; (4) removing a sialic acid, and then drying and concentrating; and (5) usingmatrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze the N-carbohydrate chain. Operation is simple and rapid, and the method can be widely applied to the analysis of the N-carbohydrate chain of the glycoproteins from different sources. An analysis result is stable and reliable, and an application prospect is wide.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for analyzing glycoprotein N-sugar chains. Background technique [0002] The N-sugar chains connected to glycoproteins are molecules formed by linking monosaccharides such as glucose, galactose, mannose, N-acetylglucose, N-acetylgalactose, and sialic acid through glycosidic bonds. N-sugar chains are catalyzed by glycosyltransferases on the surface of the endoplasmic reticulum, and are linked to the asparagine residues on newly generated proteins through N-glycosidic bonds, and enter the endoplasmic reticulum and Golgi for a series of subsequent processing. N-glycosylation usually occurs in the conserved amino acid sequence N-X-T / S (X≠P), which can be divided into high mannose type, hybrid type and compound type according to the extension of sugar chains around the pentasaccharide core. , different sugar chain structures each play an important regulatory function. A large nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/06G01N30/88G01N2030/8836
Inventor 张勇
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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