Amide monolithic column for enriching glycopeptide based on hydrophilic interaction mechanism and preparation and application method thereof

A technology of hydrophilic interaction and monolithic column, which is applied in the preparation of test samples, chemical instruments and methods, chemical/physical processes, etc., can solve problems such as synthesis process or cumbersome operation process, achieve stable and uniform structure, and improve selectivity , Preparation is simple and fast

Active Publication Date: 2014-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, many research groups have developed different types of materials based on hydrophilic interaction chromatography, including commercial agarose gel particles, hydrophilic polymer microspheres, and surface hydrophilic modified magnetic spheres, etc. (Selman, M.H., McDonnell, L.A., Palmblad, M., Ruhaak, L.R., Deelder, A.M., Wuhrer, M., Anal. Chem. 2010, 82, 1073–1081. Yu, L. Li, X. Guo, Z. Zhang, X. Liang, X.Chem.Eur.J.2009,15,12618-12626; Yeh, C.H.Chen, S.H.Li, D.T.Lin, H.P.Huang, H.J.Chang, C.I.Shih, W.L.Chern, C.L.Shi, F.K.Hsu, J.L.J.Chromatogr. A2012, 1224, 70-78;), the synthesis process or operation process of these materials is relatively cumbersome, and the enrichment of glycopeptides by offline operation will bring inevitable sample loss and time consumption

Method used

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  • Amide monolithic column for enriching glycopeptide based on hydrophilic interaction mechanism and preparation and application method thereof
  • Amide monolithic column for enriching glycopeptide based on hydrophilic interaction mechanism and preparation and application method thereof
  • Amide monolithic column for enriching glycopeptide based on hydrophilic interaction mechanism and preparation and application method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Preparation of amide-type monolithic column

[0029] Such as figure 1 As indicated, weigh 11.5mg of acrylamide, 22.2mg of N-vinylpyrrolidone and 56mg of N,N'-methylenebisacrylamide, add 420μL of dimethyl sulfoxide, shake to dissolve until clear, then add 0.9mg of azo Diisobutyronitrile, mixed until clear; add 0.15g of 1,4-butanediol and 0.3g of dodecanol, after mixing evenly, pass through N 2 30 seconds to remove O from the solution 2 , The solution was poured into a 12 cm long ultraviolet transparent quartz capillary, and both ends were sealed with silica gel. Under the irradiation of 365nm ultraviolet light, react for 15min. Take out the capillary, and pass through acetonitrile for about 1 hour to remove unreacted substances, thereby preparing an amide-type monolithic column.

[0030] 2. Internal characterization of monolithic column materials

[0031] Scanning electron microscope picture as figure 2 As shown, the prepared monolithic column has a stable and ...

Embodiment 2

[0033] 1. Standard protein sample pretreatment

[0034] Weigh 1mg of human serum immunoglobulin (IgG) and dissolve it in 1mL of 20mM ammonium bicarbonate solution, heat the reaction at 90°C for 10min, cool to room temperature, then add 4μL of 1M dithiothreitol solution (prepared with 20mM ammonium bicarbonate solution) , heated at 56°C for 1.5 h; after cooling to room temperature, 8 μL of 1M iodoacetamide solution (prepared with 20 mM ammonium bicarbonate solution) was added, and reacted at room temperature for 40 min in the dark. After the reaction, add 40 μL of 1 mg / mL trypsin solution (prepared with 20 mM ammonium bicarbonate solution), heat the reaction at 37 ° C for 20 h, then add an appropriate amount of formic acid (FA) to make the pH of the solution around 2-3 to terminate the reaction, and finally A 1 mg / mL standard proteolysis product was obtained.

[0035] A C18 pre-column was used to desalt and lyophilize the above enzymatic hydrolyzate, and finally the sample was...

Embodiment 3

[0041] 1. Selective enrichment of glycopeptides under the interference of BSA hydrolyzate

[0042] Mix the prepared 1 mg / mL IgG and BSA hydrolyzate (dissolved in 80% ACN containing 0.1% FA) at a mass ratio of 1:1 and 1:100000, cut off a 10 cm amide-type monolithic column, and load 2 μL of the above standard The mixed solution of proteolysis products was washed with 80% ACN (containing 0.1% FA) for 30 minutes, and then the glycopeptides were eluted for 10 minutes under the condition of 70% ACN (containing 0.1% FA), and the eluted enriched products were collected. Direct MALDI-TOF MS analysis.

[0043] 2. MALDI-TOF MS analysis

[0044] 1 μL of the enriched product and 1 μL of LDHB matrix (20 mg / mL DHB dissolved in 60% acetonitrile solution containing 0.1% trifluoroacetic acid) were sequentially spotted on the MALDI target plate, and the mass spectrometric identification was carried out after the sample spot was dried. Such as Pic 4-1 , 4-2 and Figure 5-1 , 5-2 As shown, ...

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Abstract

The invention relates to an amide monolithic column specially-used for enriching glycopeptide based on hydrophilic interaction mechanism and a preparation and an application thereof. The monolithic column is prepared by the steps of that: employing a monomer with an amide functional group and a cross-linking agent; adding a pore forming agent and an initiator with proper ratios; and preparing the monolithic column in a ultraviolet transparent quartz capillary in a manner of rapid photopolymerizaiton. The monolithic column is easy and quick to prepare, stable and uniform in structure, good in hydrophilic performance and has multiple amide functional groups. With the monolithic column, an on-column enrichment operation of glycopeptide based on the hydrophilic interaction mechanism in a microscale biological sample can be achieved. The monolithic column has an excellent practical value and a good application prospect in the preprocessing of glycoprotein of a biological sample and the construction of an on-line analysis platform of glycoprotein.

Description

technical field [0001] The invention relates to a glycopeptide enrichment device, that is, an amide-type monolithic column for enriching glycopeptides based on a hydrophilic interaction mechanism and its preparation and application in biological sample glycoprotein pretreatment technology. Background technique [0002] In the research field of proteomics, the influence of protein composition and structure on its function has been paid attention to, especially for post-translationally modified proteins, different modification sites and groups have different effects on protein function. However, due to its low abundance and high complexity, it has become a research hotspot and difficulty in proteomics. [0003] Glycoproteins are a class of biologically important proteins that play an indispensable role in biological processes such as cell trafficking, signal transduction, immune regulation, and biological stress, and are also biomarkers and clinical treatments for some disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/285B01J20/30B01D15/22B01D15/08G01N1/40
Inventor 张丽华蒋好袁辉明梁玉杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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