Method for selective enrichment and identification of N-linked glycopeptide

A sugar-linking and selective technology, applied in the field of selective enrichment of N-linked glycopeptides, can solve the problems of reducing the efficiency of amino chemical methods and the interference of amino microspheres enriching glycopeptides, achieving high enrichment specificity and improving Identify the effect of coverage and material availability

Inactive Publication Date: 2017-03-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, there is a significant problem in the amino chemical method, that is, the terminal amino group and side chain amino group of the peptide in the sample can also form a Schiff base with the aldehyde group on the oxidized sugar chain, and be reduced by the added reducing reagent. It will cause greater interference to the enrichment of glycopeptides by amino microspheres ( figure 1 inset in ), greatly reducing the efficiency of amino chemistry

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  • Method for selective enrichment and identification of N-linked glycopeptide
  • Method for selective enrichment and identification of N-linked glycopeptide
  • Method for selective enrichment and identification of N-linked glycopeptide

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Embodiment 1

[0038] Verify the feasibility of the described amino chemistry based on two reductive amination reactions:

[0039] Human immunoglobulin (IgG) is a simple and easy-to-obtain glycoprotein, and bovine serum albumin (BSA) is a commonly used standard non-glycoprotein. N-glycopeptides in the peptide mixture of the separation of the two are enriched by using the amino chemical method based on two reductive amination reactions of the present invention. The experimental procedure is as follows:

[0040] 1. Dissolve 200 μg of IgG and BSA at a molar ratio of 1:1 in 96 μL of 100 mM TEAB buffer (pH 8.0), heat inactivate in boiling water for 10 min, cool to 25°C, add 4 μg of trypsin, and place in a 37°C water bath Medium enzymatic hydrolysis for 12h;

[0041] 2. Add 20 μL of 4wt% CH to the resulting peptide solution 2 O / H 2 O and freshly prepared 20 μL of 0.6M NaBH 3 CN aqueous solution, react in a shaker at 25°C for 1h;

[0042] 3. Add 30 μL of 8wt% ammonia water to quench the label...

Embodiment 2

[0050] We used the amino chemistry method based on two reductive amination reactions and the amino chemistry method based on one reductive amination reaction to analyze the N-glycoproteome information in human liver cancer serum, and compared the two methods to identify Results of N-glycosylation sites.

[0051] Human liver cancer serum used in this experiment was provided by the Second Affiliated Hospital of Dalian Medical University (Dalian, China). The acquisition and use of the samples were completely legal and complied with the relevant regulations of the ethics committee of the hospital. The experimental procedure for serum samples using amino chemistry based on two reductive amination reactions is as follows:

[0052] 1. Take 15 μL (containing about 1 mg of protein) serum, dilute it to 92 μL with a buffer (pH 8.0) containing 8M Urea and 100 mM TEAB, add 8 μL of 1M DTT, and react in a water bath at 60°C for 1 hour to open the disulfide bond, then Add 0.74 mg of IAA, an...

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Abstract

The invention relates to a method for selective enrichment and identification of N-glycosylation sites in a biological sample based on a reductive amination reaction of amino and aldehyde groups and application of the method to proteomic analysis of N-glycoprotein. The method comprises the following steps: taking a biological sample containing glycoprotein, carrying out enzymatic hydrolysis by using trypsin and then blocking the terminated amino group and side-chain amino group of a peptide fragment by using formaldehyde; then oxidizing a sugar chain on glycopeptide by using a sodium periodate solution so as to produce an aldehyde group; then enriching oxidized glycopeptide with amino microspheres and carrying out treatment with peptide glycosidase PNGase F; and carrying out mass spectrometry on released N-glycopeptide so as to obtain proteome information of N-glycoprotein in the sample. The method can be used for proteomic analysis of glycosylation and can acquire identification results of corresponding glycoprotein, glycopeptide and glycosylation sites at the same time. The method is simple and highly efficient and has high throughput in identification of N-glycosylation sites on glycoprotein.

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 selectively enriching N-linked glycopeptides based on amino chemistry and its application. Background technique [0002] In eukaryotes, protein glycosylation, as one of the most common and important post-translational modifications, is involved in apoptosis, immune response, cell-cell interaction, ligand-receptor interaction and the occurrence and development of diseases Wait for the life process. At present, most of the clinically used disease markers are glycosylated proteins, such as the marker protein of liver cancer - alpha-fetoprotein (AFP), the marker of malignant tumor - cancer antigen 125 (cancer antigen 125), and the marker of prostate cancer - Prostate cancer-specific antigen (PSA), etc. Therefore, a comprehensive and in-depth study of glycoproteins is of great significance. [0003] In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N1/28
CPCG01N33/68G01N1/28
Inventor 邹汉法张章叶明亮
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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