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Enrichment method of thiosulfated peptides

An enrichment and polyamino technology, which is applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems such as the lack of omics enrichment methods, and achieves wide application, increased bonding capacity, and simple operation. fast effect

Active Publication Date: 2017-06-16
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the enrichment of sulfhydryl modification is mainly concentrated at the protein level, and there is no large-scale omics enrichment method (Angew.Chem.Int.Ed, 2014, 53, 575-581; Sci.Signaling, 2009, 2, ra72 )

Method used

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  • Enrichment method of thiosulfated peptides
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  • Enrichment method of thiosulfated peptides

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Preparation of a water-soluble polymer compound that selectively reacts with a thiol-containing polypeptide: Weigh 3 mg of iodoacetic acid-N-succinamide ester, dissolve it in a mixed solution of 400 μL methanol and 200 μL pH 8.0 phosphate buffered saline, and configure it as a reaction solution A; Weigh 16mg of aminoethanol dendrimer (butanediamine core, 6th generation), the molecular weight is 58000Da, and add in the reaction solution A, after mixing, shake at room temperature and react in the dark for 3 hours, and the reaction product is placed on the ultrafiltration membrane , with a molecular weight cut-off of 10,000Da, centrifuged at 16,000rpm for 40min to remove unreacted iodoacetic acid-N-succinamide ester, and then washed twice with phosphate buffered saline to produce a water-soluble polymer compound 1 that selectively reacts with thiol-containing polypeptides , the preparation flow chart is as figure 1 (A) shown.

[0022] Weigh 3mg of transferrin, dilute it t...

Embodiment 2

[0024] Preparation of a water-soluble polymer compound that selectively reacts with a thiol-containing polypeptide: Weigh 10 mg of iodoacetic acid-N-succinamide ester, dissolve it in a mixed solution of 480 μL methanol and 120 μL pH 7.5 phosphate buffered saline, and configure it as a reaction solution A; Weigh 16mg of aminoethanol dendrimer (butanediamine core, 6th generation), molecular weight is 58000Da, and add in the reaction solution A, after mixing, shake at room temperature and avoid light for 10 hours, and the reaction product is placed on the ultrafiltration membrane , with a molecular weight cut-off of 10,000Da, centrifuged at 16,000rpm for 40 minutes to remove unreacted iodoacetic acid-N-succinamide ester, and then washed twice with phosphate buffered saline to prepare a water-soluble polymer compound 2 that selectively reacts with thiol-containing polypeptides , the preparation flow chart is as figure 1 (A) shown.

[0025] Take 200 μg of HeLa cells to extract pro...

Embodiment 3

[0027] Weigh 3 mg of iodoacetic acid-N-succinamide ester, dissolve it in a mixed solution of 400 μL methanol and 200 μL pH 8.0 phosphate buffered saline, and configure it as reaction solution A;

[0028] Take by weighing 16mg aminoethanol dendrimers (butylene diamine core, 4th generation) (molecular weight is 30000Da), and add in the reaction solution A, make the water-soluble polymer compound that selectively reacts with mercapto-containing polypeptide, then It was used for the enrichment of thiothiol peptides in protein hydrolyzates extracted from HeLa cells, and a total of 1,200 thiol peptides were identified, indicating that this method has excellent enrichment capabilities for thiol peptides.

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Abstract

The invention relates to an enrichment method of thiosulfated peptides, and applications thereof. According to the enrichment method, a polyaminodendritic polymer is taken as a carrier, covalent modification with iodoacetic acid-N-succinamide is carried out so as to prepare a water soluble high-molecular compound capable of realizing selective reaction with mercapto peptides; the water soluble high-molecular compound is mixed with a protein enzymatic hydrolysate fully, an obtained product is placed on an ultrafiltration membrane, selective blocking of the mercapto peptides on the ultrafiltration membrane is realized via high speed centrifugation; and at last, a disulfide bond reducing agent is added onto the ultrafiltration membrane to realize selective release of the thiosulfated peptides from the high-molecular compound at a certain temperature; and the thiosulfated peptides are obtained via high speed centrifugation. Operation of the enrichment method is simple; flux is high; selectivity is high; and the enrichment method is used for large-scale enrichment of the thiosulfated peptides in complex proteome samples.

Description

technical field [0001] The invention relates to a method for enriching thiothiolated polypeptides and its application in large-scale analysis of thiothiolated polypeptides in proteome samples such as cells, tissues and body fluids. Background technique [0002] A large number of research results have shown that oxidative stress plays a key pathological role in many major diseases related to aging. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) attack lipid macromolecules, protein molecules and DNA molecules, etc., causing the disorder of cell physiological functions, further leading to cell apoptosis and necrosis. Sulfur-containing amino acid residues in proteins, including cysteine ​​(Cys) residues and methionine (Met) residues, are important sites for reactive oxygen species to play an important role in oxidation-reduction regulation. After reactive oxygen species bind to these amino acid residues, an oxidation-reduction-dependent post-translational mod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K1/16
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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