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Method for identifying N-linked oligosaccharide structure of novel erythropoiesis stimulating protein

An erythrocyte and oligosaccharide technology, which is applied in the field of identifying N-linked oligosaccharide structures of new erythropoiesis-stimulating proteins, and can solve the problems of difficulty in identifying structures, complex N-linked oligosaccharide structures, and high sialylation.

Active Publication Date: 2016-03-02
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide an N-linked oligosaccharide for identifying new erythropoiesis-stimulating proteins in order to overcome the defects that the N-linked oligosaccharides on the new erythropoiesis-stimulating protein are complex in structure, highly sialylated, and difficult to identify the structure. sugar structure method

Method used

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  • Method for identifying N-linked oligosaccharide structure of novel erythropoiesis stimulating protein
  • Method for identifying N-linked oligosaccharide structure of novel erythropoiesis stimulating protein
  • Method for identifying N-linked oligosaccharide structure of novel erythropoiesis stimulating protein

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Embodiment 7

[0050] Embodiment 7 is carried out on Agilent (Agilent) 1260 high performance liquid chromatography instrument.

Embodiment 9

[0051] Example 9 is in Beckman Coulter (Beckman_Coulter) P / ACE TM MDQ was performed on a high-efficiency capillary instrument.

Embodiment 1

[0052] Example 1 Pretreatment of new erythropoiesis-stimulating protein samples

[0053] Ultrafiltration to remove salt and surfactant: prepare a 1.5mL ultrafiltration tube washed with water in advance, load 100μL of a new erythropoiesis-stimulating protein aqueous solution with a sample protein content of 100μg, and centrifuge at a centrifugal force of 14,000g for 15min to reduce the sample volume to 1 / 10. Add water to dilute to the original volume and centrifuge again at 14000g for 15min. Then dilute and centrifuge again, the conditions are the same as the previous two times. Afterwards, the ultrafiltration tube was inverted, and the protein was recovered by centrifugation at a centrifugal force of 2000 g for 5 minutes. The recovery rate was 95%. The volume of the recovered protein sample containing water was 10 μL, which was lyophilized for use.

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Abstract

The present invention discloses a method for identifying the N-linked oligosaccharide structure of novel erythropoiesis stimulating protein. The method comprises: (1) determining the linking way of the terminal sialic acid on the N-linked oligosaccharide structure of novel erythropoiesis stimulating protein by combining sialidase digestion and laser-induced fluorescence capillary electrophoresis separation; (2) attributing the desialylated N-linked oligosaccharide structure by combining exglycosidase digestion sequencing and laser-induced fluorescence capillary electrophoresis separation; and (3) analyzing the N-linked oligosaccharide structure by using laser-induced fluorescence capillary electrophoresis and the two-dimensional separation technology of weak anion exchange chromatography and combining sialidase digestion. According to the present invention, the method has characteristics of high sensitivity, high separation degree, simple operation, low cost and high accuracy, is suitable for high-throughput detection and industrial applications, and performs the structure identification on the saccharide chain accounting for 95% by mass of the N-linked oligosaccharide structure on the novel erythropoiesis stimulating protein.

Description

technical field [0001] In particular, the present invention relates to a method for identifying N-linked oligosaccharide structures of novel erythropoiesis-stimulating proteins. Background technique [0002] Glycosylation is a form of post-translational modification that covalently links sugar chains of different structures to specific sites in protein amino acid sequences. Glycosylation can be divided into four types according to the connection method: N-glycan (N-glycan, N-linked oligosaccharide), O-glycan (O-glycan, O-linked oligosaccharide), glycophospholipid anchor (glycophospholipidanchor, GPIAnchor) and C-glycosylation. An important feature of protein glycosylation is heterogeneity, that is, a series of structurally related sugar chains (micro-heterogeneity) will be generated during the glycosylation process, and different glycosylation sites in the same glycoprotein will be connected differently. Different sugar chains (dot heterogeneity). This is because the occu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N30/02
Inventor 康经武李凤
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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