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Preparation of protein solid-phase alkylation reagent, solid-phase alkylation reagent and application

An alkylation reagent and solid-phase alkane technology, applied in the field of protein solid-phase alkylation reagents, can solve the problems of difficult to achieve deep coverage analysis of proteome, difficult to remove, unable to use strong protein extraction reagents, etc. The effect of flux, improving hydrophilicity, and shortening modification time

Active Publication Date: 2018-10-26
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

[0003] However, since this method is difficult to remove during sample processing, strong protein extraction reagents (such as surfactants such as SDS) cannot be used, so it is difficult to achieve deep coverage analysis of the proteome

Method used

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  • Preparation of protein solid-phase alkylation reagent, solid-phase alkylation reagent and application
  • Preparation of protein solid-phase alkylation reagent, solid-phase alkylation reagent and application
  • Preparation of protein solid-phase alkylation reagent, solid-phase alkylation reagent and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Modification of ATRP initiator: Weigh 4g of silica gel, add 60mL of toluene and 1.2mL of 3-(trimethoxysilylpropyl)-2-bromo-2-methylpropyl ester, mix well, stir and reflux for 90 ℃ reaction 12h. After the reaction was completed, it was washed 3 times with absolute ethanol.

[0028] 2. Modification of epoxy groups: Weigh 1.2g of silica gel particles modified with ATRP initiator, add 28mL methanol, 2.65mL glycidyl methacrylate (GMA), 0.0314mL N,N,N',N",N ”’-Pentamethyldiethylenetriamine, after mixing well, after passing through nitrogen for 5min, add 10mg CuCl and 1.7mg CuCl 2 , Sealed and refluxed at 60°C for 6h to prepare Si-GMA particles. After the reaction is completed, wash with methanol and water three times respectively, then add supersaturated EDTA solution to wash until colorless, then wash with water and methanol, and dry it for later use.

[0029] 3. PEI modification: Disperse the prepared Si-GMA particles in 50mM phosphate buffer solution (pH 8.0), add 1....

Embodiment 2

[0032] 1. Modification of ATRP initiator: Weigh 10g of silica gel, add 150mL of toluene and 3mL of 3-(trimethoxysilylpropyl)-2-bromo-2-methylpropyl ester, mix well, stir and reflux at 90°C Reaction 12h. After the reaction was completed, it was washed 3 times with absolute ethanol.

[0033] 2. Modification of epoxy groups: Weigh 3g of silica gel particles modified with ATRP initiator, add 50mL methanol, 5mL glycidyl methacrylate (GMA), 0.1mL N,N,N',N",N"' -Pentamethyldiethylenetriamine, after mixing well, add 50mg CuCl and 8.5mg CuCl after flowing nitrogen for 5min 2 , Sealed and refluxed at 60°C for 6h to prepare Si-GMA particles. After the reaction is completed, wash with methanol and water three times respectively, then add supersaturated EDTA solution to wash until colorless, then wash with water and methanol, and dry it for later use.

[0034] 3. PEI modification: Disperse the prepared Si-GMA particles in 50mM phosphate buffer solution (pH 8.0), add 1.0g PEI to a final ...

Embodiment 3

[0037] Other conditions are the same as in Example 1, and the ATRP catalyst becomes FeCl 2 / FeCl 3 , to prepare protein solid-phase alkylation reagent, which is reagent 3

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Abstract

The invention relates to a protein solid-phase alkylation reagent and its preparation and application. Using silica gel particles as a carrier, epoxy groups are modified on the surface of the carrier through atom free transfer polymerization, and then dendrites are modified accordingly by covalent bonding. The hydrophilic compounds polyethyleneimine and iodoacetic acid-N-succinamide ester were prepared as solid-phase alkylation reagents. The reagent can selectively react with the sulfhydryl group on the protein, and has the advantages of high stability, simple operation and rapid reaction. Compared with traditional alkylation reagents, using this reagent, proteins can be rapidly separated from other small molecules (such as sugars, salts, surfactants, lipids, etc.), improving the purity of proteins and significantly reducing the complexity of samples. Due to the good hydrophilicity of the surface of the reagent, the recovery rate of protein or enzymatic hydrolysis product can be significantly improved. Therefore, this reagent is suitable for the selective reaction and pretreatment of sulfhydryl groups in proteins extracted from cells, tissues, body fluids, and hair.

Description

technical field [0001] The invention relates to a protein solid-phase alkylation reagent, which can be applied to the selective reaction and pretreatment of sulfhydryl groups in protein extracted from cells, tissues, body fluids, hair, and the like. Background technique [0002] In clinical practice, in order to diagnose tumors, it is usually necessary to puncture the patient to obtain tissue samples, and then conduct pathological diagnosis by electron microscope after sectioning. This method not only takes a long time (1-4 days), but also has the risk of false positives. There has been widespread interest in classifying tissue morphology using molecular typing methods, currently mainly through mRNA transcript expression profiling or metabolomics approaches. Recently, Ruedi's research group combined pressure cycle technology and SWATH technology to develop a rapid and reproducible proteome analysis method for analyzing micro-tissue samples. This method was used to analyze 18...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F283/00C08F220/32C08F292/00C07K1/14
Inventor 张丽华袁辉明朱旭东杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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