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Universal method for enriching glycopeptides by metallic oxide

A general method and oxide technology, applied in the field of biochemical analysis, can solve the problems of insufficient versatility and unsuitability of the method, and achieve the effects of high glycosylation coverage, easy repeatability, and simple operation

Inactive Publication Date: 2013-05-15
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method can achieve highly selective enrichment of glycopeptides, but different glycopeptides require different washing and elution conditions, resulting in insufficient versatility of the method and not suitable for unknown and complex samples

Method used

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  • Universal method for enriching glycopeptides by metallic oxide
  • Universal method for enriching glycopeptides by metallic oxide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Put 1 mg of titanium oxide into a gel tip, load 10 μL of transferrin hydrolyzate (see Preparation of sample solution) (pH 3), and then elute twice with 30 μL of 0.25% ammonia solution (pH 3). 10); finally, eluted twice with 30 μL of 10% ammonia solution (PH=12) by volume to obtain the glycopeptide. The enriched glycopeptides were analyzed by mass spectrometry.

[0025] Depend on figure 1 It can be seen that the glycopeptides in the hydrolyzate of transferrin can be specifically enriched and purified by titanium oxide.

Embodiment 2

[0027] Adjust the operation mode of enrichment to centrifugation, put 1 mg of titanium oxide into a centrifuge tube, mix with 5 μL of peptiglobulin hydrolyzate (pH 3), incubate for 5 minutes, collect the precipitate after centrifugation; then use 30 μL of the volume concentration of the precipitate to be 10% CH 3 CN / 0.1%NH 4 OH (pH 10) was incubated for 5 min, and the precipitate was collected. Incubate the precipitate with 30 μL of 0.25% ammonia solution (PH=10) for 5 min, collect the precipitate after centrifugation, repeat the incubation and centrifugation steps twice; finally incubate the precipitate with 30 μL of 10% ammonia solution (PH=12) After centrifugation for 5 minutes, the supernatant was collected to obtain glycopeptides. Each supernatant was analyzed directly on a MALDI-TOF mass spectrometer.

[0028] Depend on figure 2 It can be seen that the glycopeptides in the fetuin hydrolyzate are enriched and purified by titanium oxide anisotropy.

Embodiment 3-6

[0030] Adjust the weight of the metal oxide material to 2 mg, 3 mg, 6 mg, and 10 mg. Other conditions are the same as in Example 1. The glycopeptide obtained after enrichment is subjected to mass spectrometry analysis. The experimental results show that 1 mg of the material can be effectively retained in the extraction mode operation mode. and enriched glycopeptides in glycoprotein standard digests.

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Abstract

The invention relates to the field of biochemical analysis, and discloses a method for enriching glycopeptides. In the method, metal oxide is taken as an enrichment material, and by optimization of loading, drip washing and elution conditions, the selective enrichment of the glycopeptides is realized. The method has the advantages of high selectivity of glycopeptides, wide glycosylation coverage, wide universality, simplicity in operation, excellent repeatability and so on, is suitable for selective enrichment of the glycopeptides in complex systems, and has wide application prospect in the fields of posttranslational modification proteomics research and the like in combination with mass spectrometry.

Description

technical field [0001] The invention relates to the field of biochemical analysis, in particular to the application of a metal oxide in enriching glycopeptides. Background technique [0002] Protein glycosylation is one of the most common post-translational modification processes. More than 50% of proteins are glycosylated. Protein glycosylation is involved in many biological processes such as cellular immunity, receptor activation, and signal transduction. Studies have shown that the abnormality of glycoprotein is closely related to some diseases. In addition, some glycoproteins have been developed into drugs due to their special biological functions. Therefore, in-depth research on glycoproteins can better understand and explain life phenomena, reveal the occurrence process of diseases, and play an important role in the early diagnosis of diseases and the development of new target drugs. However, due to the very low abundance of glycopeptides in the hydrolyzate after e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/40G01N1/34
Inventor 李秀玲闫竟宇梁鑫淼
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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