Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner

A quantitative analysis and sugar chain technology, applied in the analysis of materials, material analysis by electromagnetic means, measurement devices, etc., can solve the problems of loss of sialic acid information, masking of sugar chain structure information, etc., to improve the comprehensiveness and quantitative accuracy. sexual effect

Active Publication Date: 2014-09-03
BEIJING NANOPEP BIOTECH CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have their own advantages for the quantification of sugar chains, but all of them have the disadvantage of losing sialic acid information except for the pan-methylation method; and the pan-methylation method has many sugar chains because all hydroxyl groups are methylated. The structural information of the

Method used

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  • Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner
  • Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner
  • Method for relatively quantitatively analyzing carbohydrate chain in two-end labeling manner

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 sample protein

[0029] Preparation of cell protein: Extract cell protein with a cell protein reagent extraction kit (such as T-PER, RIPA), use as little lysate as possible to ensure a protein concentration of 2-10 mg / mL, and centrifuge at 14,000g for 5 minutes to remove cell residues. The protein concentrations of the two cell extracts were determined with a BCA kit (guaranteed at least three repetitions).

[0030]Serum protein preparation: Serum samples were centrifuged at 12,000g for 15 minutes, and the middle liquid part was taken. Add to a 10KD ultrafiltration tube, place the ultrafiltration tube in a matching centrifuge tube, centrifuge at 12,000g for 15min, add 400μl of 40mM NH 4 HCO 3 , centrifuge again, and repeat. Invert the filter membrane into a new centrifuge tube, centrifuge at 9000g for 3min, collect the isolated protein (about 50μl), quantify it with the Bradford method, and finally adjust the volume to 2mg / ml.

Embodiment 2

[0031] Example 2 Labeling and analysis of sugar chain ends

[0032] (1) Acethydrazide modification and release of sugar chain sialic acid

[0033] Take 2mg of the protein extracted in Example 1 and add it to a 10KD ultrafiltration membrane, centrifuge at 14000g to concentrate to about 50μL, add 300μL of 8M urea, mix well, and centrifuge at 14000g for 15min. Add 200 μL of 8M urea solution to the ultrafiltration tube, and centrifuge at 14000 g for 15 min. Discard the effluent in the collection tube, add 150 μL of 10 mM DTT (dithiothreitol) and mix well, incubate at 56 ° C for 45 min, centrifuge at 14000 g for 10 min, add 150 μL of 20 mM IAM (iodoacetamide) and mix well, dark Incubate at 20min, centrifuge at 14000g for 10min. Add 150 μL of ultrapure water to the ultrafiltration tube, centrifuge at 14,000 g for 10 min, and repeat 3 times. Through the above method, the protein can be desalted, and at the same time, the denaturation and modification of the sugar chain can be real...

Embodiment 3

[0043] Example 3 N-linked sugar chain modification and mass spectrometry analysis of standard glycoprotein RNase B

[0044] For the modification of RNase B sugar chains, the glycoprotein RNase B is firstly modified with the amidating reagent acetylhydrazide, then the glycosidase PNGase F is used to enzymatically release the sugar chains, and then the reducing end of the N-linked sugar chains is treated with the aminating reagent aniline. Modified, the obtained sugar chain was identified by MALDI-TOF, and the [M+Na] of RNase B was obtained + sugar chain peak spectrum. At the same time, the sugar chains of RNase B released without any sugar chain modification were compared, and the results were as follows figure 2 shown, from figure 2 In -A, it can be seen that the sugar chain spectrum of RNase B is fully identified. From the spectrum, it can be seen that the sugar chains of RNase B on the same glycosylation site are slightly heterogeneous, and there are 5 high-mannose glyco...

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Abstract

The invention discloses a method for relatively quantitatively analyzing a carbohydrate chain in a two-end labeling manner, and belongs to the field of quantitative carbohydrate analysis. Amidation on sialic acid and amination on a reduction end of a carbohydrate chain structure are respectively modified by utilizing a hydrazide agent and an amination agent, meanwhile, a sialic acid group of the carbohydrate chain structure is protected, and the reduction end of the carbohydrate chain is labeled by isotope, so that the complete structure information and quantitative information of the carbohydrate chain are simultaneously analyzed, and the comprehensiveness of analysis and the accuracy of the quantitation on the carbohydrate chain are improved.

Description

technical field [0001] The invention relates to a method for relatively quantitative analysis of sugar chains with two-terminal labels, belonging to the field of quantitative analysis of sugars. Background technique [0002] Carbohydrates are a class of macromolecular substances that exist in large quantities in biological organisms, and play an important role in various processes of organisms. The main forms of carbohydrates are carbohydrate complexes, such as glycolipids, glycoproteins, proteoglycans, glycosaminoglycans, and lipopolysaccharides. Among them, glycosylated proteins are widely distributed on the cell surface and in the extracellular matrix, and the sugar chain structure on them is related to many important biological functions, mainly including regulating the conformation and stability of proteins, and controlling the half-life of proteins and even cells. In addition, the specific binding of these sugar chain structures as ligands mediates targeted recognitio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64
Inventor 关锋杨刚龙谭增琦陆微庞星辰
Owner BEIJING NANOPEP BIOTECH CORP LTD
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