Quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and application of quantitative method in hepatoma marker screening

A technology of sialylation and glycoprotein, applied in measuring devices, material analysis by electromagnetic means, instruments, etc., to achieve the effect of easy-to-obtain materials, easy operation, and high enrichment specificity

Inactive Publication Date: 2016-10-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

These reported methods directly compare the difference of sialylation at the same glycosylation site between disease samp...

Method used

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  • Quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and application of quantitative method in hepatoma marker screening
  • Quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and application of quantitative method in hepatoma marker screening
  • Quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and application of quantitative method in hepatoma marker screening

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Embodiment 1

[0025] Example 1 Occupancy of N-linked sialylated sugar chains on quantitative standard glycoproteins

[0026] Human serum transferrin (HT) is a simple and easy-to-obtain glycoprotein, and the sugar chain ends on its two N-glycosylation sites are all sialic acid (document 7.Satomi, Y., Shimonishi, Y .,Hase,T.&Takao,T.Site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography / electrospray ionization mass spectrometry.Rapid communications in mass spectrometry:RCM18,2983-2988,doi:10.1002 / rcm.1718( 2004).). The terminal sialic acid can be partially removed by desialidase to obtain desialylated transferrin (AHT) (Reference 6). The experimental process for quantifying the occupancy of N-linked sialylated sugar chains on these two standard glycoproteins using the method described in the present invention is as follows: figure 1 shown.

[0027] 1. Two aliquots of 1mg each of the two standard glycoproteins were dissolved in 100μL containing 8M Urea ...

Embodiment 2

[0037] We used this method to quantify the occupancy of N-linked sialylated sugar chains on glycoproteins in the liver tissues of liver cancer patients and normal people, and compared the N-linked sialylated sugar chains on glycoproteins in the two states Changes in the occupancy rate of HCC, thereby discovering potential biomarkers in human liver cancer.

[0038] The human liver tissue samples used in this experiment were provided by the Second Affiliated Hospital of Dalian Medical University (Dalian, China), which are non-cancerous tissues around the liver (≥ 2 cm) of hepatocellular carcinoma (HCC). The normal tissue part (Normal) had been pathologically sliced, and the acquisition and use of the samples were completely legal and in compliance with the relevant regulations of the ethics committee of the hospital. Protein solutions extracted from liver tissue were processed in the same manner as standard proteins, such as figure 1 shown. Finally, the occupancy rate of 496 s...

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Abstract

The invention relates to a quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and an application of the quantitative method in hepatoma marker screening. The method comprises the following steps: two biological samples containing glycoprotein under a same state are taken, then a sodium periodate solution is used for respectively processing the two biological samples for performing differential oxidation, then a hydrazide chemical method on protein is used for enriching glycoprotein in the samples after differential oxidation, then differences of different stable isotopes can be used for labeling the glycopeptide on hydrazide microspheres, and peptide-N-glycosidase (PNGase) is used for processing, and finally mass spectrometry is carried out on mixing difference-labeled N-glycopeptide so as to quantify the occupancy of N-bond sialylated sugar chains. The method can be used for glycosylation-modified proteomics analysis, can simultaneously obtain the corresponding glycoprotein, glycopeptide and glycosylation bit identification results, and especially can be used for screening of a latent biological marker for hepatic cellular cancer(HCC). The method has the advantages of simpleness and high efficiency.

Description

technical field [0001] The invention belongs to the technical field of glycosylation proteomics in the direction of proteomics research, and specifically relates to a method for high-throughput quantification of the occupancy of N-linked sialylated sugar chains on glycoproteins by differential oxidation and labeling based on hydrazide chemistry and its application in Application in the screening of liver cancer markers. Background technique [0002] Protein glycosylation, as one of the most common and important post-translational modifications, plays an important role in protein folding, conformational stability and activity. Glycosylated proteins (glycoproteins for short) are involved in the molecular recognition of cells and cells, cells and extracellular matrix, apoptosis, protein interaction, immune response and other life processes. Therefore, abnormal changes in glycoproteins are often accompanied by the occurrence and development of many diseases. That is, abnormal ...

Claims

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Application Information

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IPC IPC(8): G01N27/62
Inventor 邹汉法张章叶明亮
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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