Isotope-labeled bionic sugar or sugar group and preparation method and applications thereof

A technology of isotope labeling and biomimetic sugar, which is applied in the fields of analytical chemistry, medicine and biotechnology, and can solve the problems of cumbersome operation, high experiment cost and differences

Active Publication Date: 2019-07-19
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009]However, the current quantitative methods of isotope labeling have their own disadvantages: enzymatic hydrolysis to introduce isotope labeling can only introduce a molecular weight difference of 2 Da, which requires additional deconvolution calculations; The introduction of isotope labels is only applicable to cell samples, and the experimental cost is high; while most of the current chemical derivatization methods for introducing isotope labels are cumbersome, all samples need to undergo the same deri

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  • Isotope-labeled bionic sugar or sugar group and preparation method and applications thereof
  • Isotope-labeled bionic sugar or sugar group and preparation method and applications thereof
  • Isotope-labeled bionic sugar or sugar group and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1. Investigation of linear relationship and coefficient of variation based on sugar chain standards and glycoprotein standards

[0128] Taking the N-sugar chain standard NA2G1F and the glycoprotein standard IgG as examples for quantitative analysis, the linear relationship of the quantitative analysis of this method is investigated, including the following steps:

[0129] 1. Preparation of standard samples

[0130] 10 μg of sugar chain standard product NA2G1F (purchased from Ludger Company, the same below) was dissolved in 200 μL of ultrapure water to prepare a storage solution with a concentration of 0.05 mg / mL.

[0131] Dissolve 1 mg of glycoprotein standard IgG (purchased from Sigma-Aldrich, Cat. No. I4506, the same below) in 200 μL of normal saline (0.85% NaCl) to prepare a stock solution with a concentration of 5 mg / mL.

[0132] 2. Enzymatic hydrolysis of sugar chains of glycoproteins

[0133] Take 20 μL of glycoprotein IgG stock solution, add 40 μL of 2%...

Embodiment 2

[0155] Example 2. Analysis of sugar chains in serum samples

[0156] In order to further verify the applicability of the quantitative method in this paper in complex biological samples, we verified the application of this quantitative method in the quantitative analysis of human serum N-glycan groups through multiple repeated analysis experiments.

[0157] 1. Serum sample collection and storage

[0158] Blood samples were collected from the Cancer Hospital Affiliated to Fudan University. All experimental operations and research contents were approved by the Ethics Committee of Cancer Hospital Affiliated to Fudan University, and written informed consent was obtained from all subjects before sample collection.

[0159] The serum separation method was carried out according to the routine operation: firstly, 5 mL of venous blood was drawn, and it was placed in a coagulation-promoting tube at room temperature for 30 minutes. After coagulation, it was centrifuged at 3,000 rpm for 1...

Embodiment 3

[0182] Embodiment 3. same day reproducibility investigation

[0183] On the same day, take a serum sample and process it as an internal standard according to the internal standard process, and take the same serum sample at the same time, and divide it into 3 parts and process it according to the sample process. The internal standard serum samples processed according to the internal standard procedure were mixed with the three samples respectively, and then mass spectrometry analysis was performed as described above.

[0184] After data analysis and processing, it can be seen that the average coefficient of variation (CV) of the 20 most abundant sugar chains is only 4.6%, which is significantly lower than the existing quantitative methods of N-glycomics (CV: 14.2%) (Vreeker, G.C.M.; Nicolardi, S.; Bladergroen, M.R.; van der Plas, C.J.; Mesker, W.E.; Tollenaar, R.; , 90(20), 11955-11961).

[0185] The results show that the quantitative method has excellent quantitative reprodu...

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Abstract

The invention discloses isotope-labeled bionic sugar or sugar group and a preparation method and applications thereof. Specifically, the isotope-labeled bionic sugar or sugar group disclosed by the invention include a reducing alcohol hydroxyl located at the reducing end of the sugar chain and an isotope label, wherein the molecular weight of each bionic sugar chain is increased by 3 Daltons compared with that of corresponding unmodified sugar chain, and the bionic sugar (group) has the same sugar chain composition and similar sugar chain abundance as the unmodified sugar (group). The invention also provides a method for analyzing the sugar group in a sample by using the isotope-labeled bionic sugar or the sugar group, and the method comprises the step: carrying out mass analysis on the mixture of the isotope-labeled internal standard bionic sugar chain and the sample sugar chain which is not labeled by the isotope so as to carry out qualitative and/or quantitative analysis on the sugar group in the sample. The method has the advantages of both the simple operation of a non-isotope-labeled glycomics method and the accuracy of an isotope-labeled glycomics method, can be used for high-throughput detection, and has wide application prospect.

Description

technical field [0001] This application belongs to the fields of biotechnology, analytical chemistry technology and medicine. Specifically, this article relates to isotope-labeled biomimetic sugars or glycans, their preparation methods and applications, especially a simple, high-throughput, and accurate stable isotope internal standard glycan analysis method. Background technique [0002] Glycosylation is a ubiquitous post-translational modification that not only affects protein structure, solubility, and stability, but also involves multiple biological processes, such as protein folding, cell recognition, and receptor-ligand binding. Studies have reported that about 50% of mammalian proteins undergo glycosylation modifications. Abnormal glycosylation of glycoproteins is closely related to many diseases, including arthritis, congenital diseases, and tumor development and metastasis. [0003] In recent years, considering the advantages of body fluid samples, people have beg...

Claims

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Application Information

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IPC IPC(8): C07H21/00C07H1/00C07B59/00G01N33/68
CPCC07H21/00C07H1/00C07B59/005G01N33/6851G01N33/6848C07B2200/05
Inventor 顾建新任士芳秦文俊
Owner FUDAN UNIV
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