Subcellular structure characteristic N-linked carbohydrate chain and application thereof

A subcellular and cellular technology, applied in the field of glycobiology, can solve the problems of lack of organelle sugar chain structure data, and the inability to reveal the overall sugar group characteristics of subcellular structure.

Active Publication Date: 2016-06-08
上海微江生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Various important forms of glycosylation in eukaryotic cells are synthesized in the endoplasmic reticulum and Golgi body. Many studies have also shown that there are complex glycoconjugates in the nucleus and cytoplasm, and they play an important role , however, apart from a few clearly described specific examples, there is still a lack of definite sugar chain structure data in organelles
Current subcellular research mainly focuses on the analysis of the glycosylation modification of a single protein, and cannot reveal the characteristics of the overall glycome of the subcellular structure

Method used

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  • Subcellular structure characteristic N-linked carbohydrate chain and application thereof
  • Subcellular structure characteristic N-linked carbohydrate chain and application thereof
  • Subcellular structure characteristic N-linked carbohydrate chain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Extraction of Cell Broken Total Protein

[0045] Place 15mL centrifuge tubes, pipettes and other test materials in a biological safety cabinet for ultraviolet sterilization for 15 minutes; take the cell culture bottle and pour out the old medium, add 1.5mL trypsin to quickly clean the bottom of the bottle, and then add 1.5mL trypsin after suction , placed in a 37°C cell incubator to react for 3 minutes, after the reaction was completed, microscopic examination ensured that the cells were fully digested and released from the wall; adding 1.5mL of cell culture medium to remove the enzyme effect, fully blowing and aspirating the bottom of the culture bottle, after mixing, aspirate and move to a 15mL centrifuge Centrifuge the tube at 1000r / min for 5min; after centrifugation, remove the supernatant, add 5mL of 1×PBS, carefully blow and aspirate to resuspend the cells for washing, then centrifuge at 1000r / min for 5min, repeat 3 times; add 1mL LT-PER to the obtained ...

Embodiment 2

[0046] Example 2: Extraction of subcellular structural proteins

[0047] The separation method of the subcellular structure adopted in the present invention is an experimental scheme established after modification on the basis of the previous separation method. cells (~1×10 7 ) was digested with trypsin, and washed twice with pre-cooled PBS (0.01M phosphate buffer saline, 0.15M NaCl, pH7.4). Add 500ul homogenate (50mMHEPESpH7.4, 3mMMgCl 2 , 1mMEGTA) on ice for ultrasound (ultrasonic time 3s, interval time 15s, working times 10 times, power 400w). After centrifugation at 720g for 15 min, the supernatant was transferred to a new tube and recorded as (S1). 500 μL of 250 mMSTM DPS buffer (250 mM sucrose, 50 mM Tris-HCl) was added to the pellet, centrifuged at 720 g for 15 min, and the supernatant was transferred to a new tube, which was recorded as (S2). Add 2MSTMDPS buffer (2M sucrose, 50mM Tris-HCl (pH7.4), 5mM MgCl 2 , 1 mMDTT, 1 mMPMSF, 25 mg / ml spermine, 25 mg / mL spermid...

Embodiment 3

[0049] Example 3: Mass spectrometric analysis of N-linked glycans of YTS1 subcellular structure

[0050] (1) Acethydrazide modification and release of sugar chain sialic acid extracted from subcellular structural protein samples by enzymatic method

[0051] After the 10kD ultrafiltration membrane is assembled into a collection tube for collecting waste liquid during the ultrafiltration process, add a sample protein solution corresponding to a volume of 1 mg of protein, fill up the liquid surface of each tube with 8M urea, and mix well. Centrifuge at 14000g for 15min, concentrate the solution to the bottom of the ultrafiltration membrane tube, discard the effluent; add 300μL of 8mol / L urea, centrifuge at 14000g for 15min, add 200μL of 8M urea, centrifuge, discard the effluent; add 150μL of 10mMDTT solution to fully Mix well and incubate at 56°C in a dry-type thermostat for 45 minutes. After the reaction, centrifuge at 14,000g in a desktop centrifuge for 15 minutes, discard the ...

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Abstract

The invention discloses a subcellular structure characteristic N-linked carbohydrate chain and the application thereof and belongs to the technical field of glycobiology. Bladder YTS1 cells are used as the experiment material, the protein components of four subcellular structures (microsome, chondriosome, cell nucleus and cytoplasm) are obtained with the differential centrifugation method, and subcellular structure difference carbohydrate chain distribution is displayed through the change of the type and structure of subcellular structure protein galactosylated modification identified through a united lectin chip and a mass spectrum (PS) respectively. Subcellular structure characteristic N-linked glycan of ten kinds of human bladder YTS1 cells is obtained. By means of characteristic N-linked glycan, indirect detection and qualitative or quantitative detection of other target substances in corresponding subcellular structures can be achieved.

Description

technical field [0001] The invention relates to a characteristic N-linked sugar chain of a subcellular structure and an application thereof, belonging to the technical field of glycobiology. Background technique [0002] Glycosylation is the most important post-translational modification of proteins, and more than half of natural proteins are modified by glycosylation. Glycosylation of proteins can promote protein folding, prevent protein hydrolysis by proteases, and guide protein transport. N-glycosylation modification of protein can regulate the localization, function, activity, lifespan and diversity of protein in cells. Thus, the sugar chains of cells can serve as new genetic codes for cell physiology. Although glycobiology is still in the early research stage, more and more studies focus on the structural and functional analysis of glycosylation. [0003] Various important forms of glycosylation in eukaryotic cells are synthesized in the endoplasmic reticulum and Gol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62
CPCG01N33/6848G01N2440/38
Inventor 杨刚龙关锋黄鲁瑜郭佳
Owner 上海微江生物技术有限公司
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