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Preparation of protein solid-phase alkylation reagent, and solid-phase alkylation reagent and applications thereof

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: 2017-05-10
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, and solid-phase alkylation reagent and applications thereof
  • Preparation of protein solid-phase alkylation reagent, and solid-phase alkylation reagent and applications thereof
  • Preparation of protein solid-phase alkylation reagent, and solid-phase alkylation reagent and applications thereof

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, add 10mg CuCl and 1.7mg CuCl after passing nitrogen gas 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.

[0029] 3. PEI modification: Disperse the prepared Si-GMA particles in 50mM phosphate buffer solution (pH 8.0), add 1.0g PEI to ...

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 present invention relates to a protein solid-phase alkylation reagent, preparation and applications thereof. According to the preparation, silica gel particles are used as a carrier, an epoxy group is modified on the surface of the carrier through atom transfer radical polymerization, and dendritic hydrophilic compounds such as polyethyleneimine and iodoacetic acid-N-succinamate are sequentially modified through covalent bonding to prepare the solid-phase alkylation reagent. According to the present invention, the reagent can selectively react with the mercapto group on proteins, and has advantages of high stability, simple operation, quick reaction and the like; compared to the traditional alkylation reagent, the protein solid-phase alkylation reagent of the present invention has the following advantages that by using the protein solid-phase alkylation reagent, rapid separation of proteins and other small molecules (such as saccharides, salts, surfactants, lipids, and the like) can be achieved, the protein purity can be improved, and the complexity of the sample can be significantly reduced; and the reagent has good surface hydrophilicity, and can significantly improve the recovery rate of proteins or hydrolyzates, such that the reagent is suitable for the selective reaction and the pretreatment of the mercapto group in proteins extracted from cells, tissues, body fluids, hairs, and the like.

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 Applications(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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