Method for enriching phosphorylation peptide fragments and proteins and application

A technology for phosphorylating peptides and proteins, applied in chemical instruments and methods, peptide preparation methods, peptides, etc., can solve problems such as instability and inability to achieve specific enrichment, and achieve faster and more adequate reactions.

Pending Publication Date: 2019-06-18
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

However, the current research on N-phosphorylation modification and S-phosphorylation modification is very insufficient, mainly because the P-N bond is unstable under acidic conditions, while the S-P bond is unstable under both acidic and alkaline conditions, making it impossible to use Traditional enrichment strategies achieve specific enrichment

Method used

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  • Method for enriching phosphorylation peptide fragments and proteins and application
  • Method for enriching phosphorylation peptide fragments and proteins and application
  • Method for enriching phosphorylation peptide fragments and proteins and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Carboxylation of amantadine, the steps are as follows:

[0051] 1) Carboxylation reaction: Suspend 1 mol of 1-adamantanamine, referred to as AD1, 1 mol of 3-bromopropionic acid and 3 mol of potassium carbonate in 20 mL of acetonitrile, and reflux the mixture for 12 h.

[0052] 2) Post-treatment: the reaction mixture was filtered through diatomaceous earth, and fully washed with acetonitrile. The filtrate was concentrated, and the residue was purified through a chromatographic column using dichloromethane and methanol as mobile phases, and evaporated to dryness to obtain a colorless solid, referred to as AD1-APPA.

[0053] 2. A phosphoric acid recognition functional molecule: a protected precursor molecule is generated by reacting N-tert-butoxycarbonyl-L-tyrosine and N,N'-lutidine amine in the presence of paraformaldehyde, referred to as Boc-dpa, after deprotection of Boc-dpa, a functional molecule is obtained, which is called dpa for short. Proceed as follows:

[...

Embodiment 2

[0081] 1. Carboxylation of amantadine, the steps are as follows:

[0082]1) Carboxylation reaction: Suspend 500 mg of 1-adamantanamine, referred to as AD1, 510 mg of 3-bromopropionic acid and 1.37 g of potassium carbonate in 20 mL of acetonitrile, and reflux the mixture for 10 h.

[0083] 2) Post-treatment: the reaction mixture was filtered through diatomaceous earth, and fully washed with acetonitrile. The filtrate was concentrated, and the residue was purified through a chromatographic column using dichloromethane and methanol as mobile phases, and evaporated to dryness to obtain a colorless solid, referred to as AD1-APPA.

[0084] 2. A phosphoric acid recognition functional molecule: a protected precursor molecule is generated by reacting N-tert-butoxycarbonyl-L-tyrosine and N,N'-lutidine amine in the presence of paraformaldehyde, referred to as Boc-dpa, after deprotection of Boc-dpa, a functional molecule is obtained, which is called dpa for short. Proceed as follows:

...

Embodiment 3

[0112] 1. Carboxylation of amantadine, the steps are as follows:

[0113] 1) Carboxylation reaction: Suspend 400mg of 1-adamantanamine, referred to as AD1, 600mg of 3-bromopropionic acid and 1.5g of potassium carbonate in 20mL of acetonitrile, and reflux the mixture for 8h.

[0114] 2) Post-treatment: the reaction mixture was filtered through diatomaceous earth, and fully washed with acetonitrile. The filtrate was concentrated, and the residue was purified through a chromatographic column using dichloromethane and methanol as mobile phases, and evaporated to dryness to obtain a colorless solid, referred to as AD1-APPA.

[0115] 2. A phosphoric acid recognition functional molecule: a protected precursor molecule is generated by reacting N-tert-butoxycarbonyl-L-tyrosine and N,N'-lutidine amine in the presence of paraformaldehyde, referred to as Boc-dpa, after deprotection of Boc-dpa, a functional molecule is obtained, which is called dpa for short. Proceed as follows:

[0116...

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Abstract

The invention relates to functional molecules for specifically recognizing phosphorylation peptide fragments and proteins in a neutral buffered solution and application. Through a reaction of N-t-butyloxycarboryl-L-tyrosine and N, N'-dipicolylamine, supporting molecules are formed, and metal ions are complexed onto the supporting molecules to form phosphoric acid recognition functional molecules;the phosphoric acid recognition functional molecules are covalently bound with carboxylated adamantane ammonium, and guest molecules with enriched groups are obtained; cucurbituril is covalently modified on the surfaces of silicon spheres, and main molecular functional materials are obtained; and finally, the phosphorylation peptide fragments and the proteins are enriched by capturing the guest molecules in the solution through main materials. The novel phosphoric acid recognition functional molecules can realize liquid phase recognition and solid phase separation of N-phosphorylated peptide fragments and the proteins under a neutral buffer system.

Description

technical field [0001] The invention relates to a functional molecule capable of specifically recognizing phosphorylated peptides and proteins in a neutral buffer solution, and a strategy for capturing the functional molecules by using host-guest interaction to enrich the phosphorylated peptides and proteins. Background technique [0002] The post-translational modification of proteins often regulates various physiological activities of living organisms, among which protein phosphorylation modification is one of the most common post-translational modifications, which involves a variety of regulatory processes in vivo, including metabolism, transcription, translation, protein degradation, in vivo Homeostasis, cell signaling, proliferation, differentiation and cell survival etc. (Nat. Biotechnol, 2005, 23, 94-101). According to the different amino acids that occur, phosphorylation modifications are divided into O-phosphorylation modifications (serine phosphorylation, threonine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22G01N33/68
Inventor 张丽华胡晔晨江波杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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