Analytical method for researching protein structure or protein-protein interaction

A protein structure and protein technology, which is applied in the analysis field of studying protein structure or protein interaction, can solve the problems of inability to realize the analysis of the whole proteome crosslinking information, difficult to realize the identification of peptide crosslinking, and many mismatches in the identification results. Identify analysis time reduction, scale reduction, and effect of scale reduction

Active Publication Date: 2017-12-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF5 Cites 25 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the shortcomings of the traditional cross-linking mass spectrometry technology that cannot realize the analysis of the whole proteome cross-linking information of complex biological samples, many mismatches in the identification results, and the difficulty in realizing the cross-linking identification of polypeptides, the present invention provides a cross-linking method with a cleavable group. The linking agent cross-links and enzymatically hydrolyzes the protein complex in the cell, and a part of the cross-linked peptide undergoes a derivatization reaction; a part of the cross-linked peptide undergoes a cross-linking agent fragmentation and peptide enrichment reaction

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Analytical method for researching protein structure or protein-protein interaction
  • Analytical method for researching protein structure or protein-protein interaction
  • Analytical method for researching protein structure or protein-protein interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Cross-linking reaction of protein samples

[0027] Dissolve 10 μg of bovine serum albumin sample (BSA) in 20 mM 4-hydroxyethylpiperazineethanesulfonic acid buffered saline solution (HEPES) at pH 7.4 to a final protein concentration of 1 mg / mL and use dimethyl sulfoxide (DMSO) Dissuccinimidyl tartrate (DST) was prepared at a concentration of 25 mM, the cross-linking agent was added to the BSA solution to make the final concentration 1 mM, and reacted at room temperature for 1 h.

[0028] 2. Removal of excess cross-linking agent

[0029] Ammonium bicarbonate solution (ABC) was added to the reaction solution in step 1 to make the final concentration 50 mM to terminate the cross-linking reaction.

[0030] 3. Dissolution, denaturation and reduction of protein samples

[0031]Add urea and DTT to the cross-linked BSA solution in step 2, so that the final concentration of urea in the solution is 8M, and the final concentration of DTT is 10mM, and react in a water bath at 3...

Embodiment 2

[0050] 1. Cross-linking reaction of BSA protein sample; removal of excess cross-linking agent; steps of dissolution, denaturation and reduction, alkylation and enzymatic hydrolysis of protein sample are the same as in Example 1.

[0051] 2. Derivatization, fractionation and mass spectrometry analysis of cross-linked peptides

[0052] An aliquot of the lyophilized BSA cross-linked hydrolyzate from step 1 was taken and redissolved with 100 mM triethylamine-carbonic acid buffer (TEAB), pH 8. After dimethylation reaction, desalting, lyophilization, samples were fractionated using cation exchange separation, desalting, lyophilizing and reconstitution using 0.1% formic acid solution for mass spectrometry analysis.

[0053] 3. Fragmentation and enrichment of cross-linked peptides, removal of non-specifically adsorbed peptides, and release of peptides are the same as in Example 1.

[0054] 4. Fractionation of enriched cross-linked peptides

[0055] The enriched cross-linked peptides...

Embodiment 3

[0063] 1. Cross-linking reaction of protein samples

[0064] 10 μg of rabbit creatine kinase protein sample (CK) was dissolved in 50 mM phosphate-buffered saline (PBS) with a pH of 7.4, and the final protein concentration was 1 mg / mL. DST was prepared at a concentration of 25 mM using dimethylformamide (DMF). The cross-linking agent was added to the CK solution so that the final concentration was 1 mM, and reacted at room temperature for 1 h.

[0065] 2. Removal of excess cross-linking agent

[0066] Tris buffer solution (Tris) was added to the reaction solution in step 1 to make the final concentration 50 mM to terminate the cross-linking reaction.

[0067] 3. Dissolution, denaturation and reduction of protein samples

[0068] Add urea and DTT to the cross-linked CK solution in step 2, so that the final concentration of urea in the solution is 8M, and the final concentration of DTT is 10mM, and react in a water bath at 37°C for 30min.

[0069] 4. Alkylation and enzymatic h...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an analytical method for researching protein structure or protein-protein interaction. The method comprises the following steps: performing crosslinking and enzymolysis on a protein composite in a cell by using a crosslinking agent with reactive groups on two sides and a breakable group, and taking a part of the enzymatic hydrolysate for a derivatization reaction for mass spectrometry; after breaking the crosslinking agent by means of a chemical method for the other part of the enzymatic hydrolysate, enriching peptide sections with an enriching material, and performing mass spectrometry on the enzymatic hydrolysate without the enriched peptide sections; determining the crosslinked peptide sections according to a library searching result so as to establish a peptide section library; finding out a candidate peptide section from the peptide section library according to N-terminal amino acid information of the crosslinked peptide section determined in the mass spectrogram of the crosslinked peptide section; and determining the crosslinked peptide section sequence by combining the mass spectrogram m/z of the crosslinked peptide section and the characteristic ions of the peptide section so as to obtain the protein structure and protein-protein interaction information. The method has the advantage of being simple to operate, and is applied to structural analysis of proteins and analysis of protein-protein composite interaction.

Description

technical field [0001] The present invention relates to an analytical method for studying protein structure or protein interaction. Background technique [0002] The spatial structure information of proteins and the interaction information between proteins are of great significance to the study of protein functions. The change of protein spatial conformation in organisms and the interaction between proteins constitute the basis of a series of important physiological activities in cells. Therefore, the study of protein spatial structure information and the mode and degree of protein-protein interaction will help to solve many problems such as the analysis of protein function, the elucidation and treatment of disease pathogenic mechanism, and the development of new drugs. However, traditional techniques for studying protein structures and interactions, such as nuclear magnetic resonance (NMR) and X-ray crystallography, have relatively high requirements for protein purity, cry...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62
CPCG01N33/6845G01N33/6848
Inventor 张丽华方菲赵群杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap