Glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and MALDI-TOF-MS analysis method

A MALDI-TOF-MS, glycoprotein technology, applied in analytical materials, material separation, measurement devices, etc., can solve the problems of complicated operation steps, sample loss, etc., to improve the response of mass spectrometry signals, increase signal intensity, and remove detection signals. the effect of interference

Active Publication Date: 2014-12-03
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing derivatization methods often require complicated steps, resulting in unnecessary sample loss

Method used

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  • Glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and MALDI-TOF-MS analysis method
  • Glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and MALDI-TOF-MS analysis method
  • Glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and MALDI-TOF-MS analysis method

Examples

Experimental program
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Effect test

preparation example Construction

[0045] a) Preparation of graphene oxide. Usually, 0.5g phosphorus flake graphite and 0.5g Na 2 NO 3 Added to 23mL pre-cooled concentrated sulfuric acid. Subsequently, 4 g KMnO was slowly added to it 4 . The resulting mixture was stirred in an ice bath for 10 min and then transferred to a 35 °C water bath for stirring. After 1 h, 40 mL of deionized water was slowly added dropwise to the above system while maintaining the temperature of the system at 90 to 95°C. After 1 h, 100 mL of deionized water and 3 mL of H 2 o 2 And the reaction solution was stirred at room temperature for 2 h. Subsequently, the resulting product was washed with 1000 mL of deionized water containing 5 mL of HCl and then with deionized water until the pH of the eluent became neutral. Finally, the obtained product was vacuum-dried at 50° C. for 24 h to obtain the desired graphene oxide.

[0046] b) the above-mentioned graphene oxide obtained with NaBH 4 Carry out reduction treatment to obtain graph...

Embodiment 1

[0047] Example 1: One-step enrichment-derivatization treatment and MALDI-TOF-MS analysis of standard oligosaccharides based on graphene

[0048] One-step enrichment and derivatization of the standard oligosaccharide maltoheptaose (DP7) based on graphene and PBH.

[0049] 10 pmol of DP7 standard, 500 pmol of PBH (PBH) and 0.1 mg of graphene were mixed in 0.5 mL of methanol containing 0.5% acetic acid, and incubated at 90° C. for 1 h. After discarding the supernatant, the obtained graphene sample was washed three times with deionized water and methanol, respectively, and the enriched sugar chains were eluted with DMF, and analyzed by MALDI-TOF-MS.

[0050] The mass spectrometry conditions are: the instrument is 4800 Proteomic Analyzer, the positive reflector mode is selected, the acceleration voltage is 20KV, the scanning range is m / z1000-4000, the laser energy is 6000, and each mass spectrogram is composed of the total number of laser emission pulses A cumulative average of 10...

Embodiment 2

[0054] Example 2: One-step enrichment-derivatization treatment and MALDI-TOF-MS analysis of standard glycoprotein N-glycan chains based on graphene

[0055]Asialo-fetuin was dissolved in 50 mM ammonium bicarbonate (pH=8.0) or phosphate buffer (pH=7.8) at a concentration of 1 mg / mL. Afterwards, heat denaturation at 95°C for 10 minutes, and after cooling, add the required enzyme at the ratio of 1 U peptide N-glycosidase F (PNGase F): 100 μg standard glycoprotein (asialofetuin), and bathe in water at 37°C for 16 hours, so that Its sugar chains are fully released. Then add an appropriate amount of PBH and graphene (wherein, the ratio of graphene and PBH is 1mg: 15nmol; the molar ratio of PBH and sugar chains is 50: 1) in the system, and the resulting mixture is contained in a mass concentration of 0.5% Incubate in acetic acid in methanol for 1 h at 90°C. After discarding the supernatant, the obtained graphene sample precipitate was washed three times with deionized water and met...

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Abstract

The invention discloses a glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and an MALDI-TOF-MS analysis method. The chain one-step enrichment-derivation processing method comprises performing enzymatic-hydrolysis release on a glycoprotein N-carbohydrate chain, using graphene and pyrenebutyric hydrazide to performing one-step enrichment and derivation on N-carbohydrate chain, eluting N-carbohydrate chain, performing mass spectrometry, and the like. By utilizing the special pi-pi conjugate interaction of an aromatic compound and graphene and utilizing the specific efficient covalent coupling reaction of an aromatic compound containing a hydrazide or amino functional group and a hemiacetal of the N-carbohydrate chain, one-step enrichment and derivation of N-carbohydrate chain is realized, and efficient derivation of N-carbohydrate chain is finished when specific enrichment is performed. Therefore, the method avoids complicated operation steps, improves sample processing flux and substantially reduces sample loss.

Description

technical field [0001] The invention relates to a graphene-based one-step enrichment-derivatization treatment of glycoprotein N-sugar chains and a MALDI-TOF-MS analysis method. Background technique [0002] More than 50% of the proteins in the human body are glycosylated. Glycosylation is involved in almost all important life processes, such as fertilization, development, immune response, intercellular recognition and communication, etc. Among them, the composition and structure of protein N-glycans have a huge impact on the conformation, function and interaction with other biomolecules of glycosylated proteins. At the same time, studies have shown that the occurrence and development of various diseases are accompanied by changes in the composition and structure of protein N-glycan chains, such as tumors, autoimmune diseases, and diabetes. Therefore, high-throughput, high-sensitivity N-glycan detection technology is particularly important for elucidating the role of sugar ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08G01N30/02
Inventor 钱小红秦伟捷白海红
Owner ACADEMY OF MILITARY MEDICAL SCI
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