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A cross-linked peptide enrichment method and its application in protein interaction research

A protein and enrichment technology, applied in the preparation of test samples, material analysis by electromagnetic means, instruments, etc., can solve the problem of inability to achieve selective enrichment of cross-linked peptides

Active Publication Date: 2021-06-08
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

[0005] In order to overcome the shortcomings of conventional chemical cross-linking methods that cannot achieve selective enrichment of cross-linked peptides and introduce modification groups on cross-linked peptides, the present invention provides a The cross-linking agent cross-links and enzymatically hydrolyzes the intracellular protein complex, and then uses boron affinity materials to selectively enrich the cross-linked peptides and release them efficiently

Method used

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  • A cross-linked peptide enrichment method and its application in protein interaction research
  • A cross-linked peptide enrichment method and its application in protein interaction research
  • A cross-linked peptide enrichment method and its application in protein interaction research

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Cross-linking reaction of protein samples

[0023] 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.

[0024] 2. Removal of excess cross-linking agent

[0025] 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.

[0026] 3. Dissolution, denaturation and reduction of protein samples

[0027] 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 ...

Embodiment 2

[0041] 1. Cross-linking reaction of BSA protein samples; removal of excess cross-linking agent; dissolution, denaturation and reduction, alkylation, enzymatic hydrolysis, enrichment of cross-linked peptides, removal of non-specific adsorption peptides and peptide The release steps of the segments are the same as in Example 1.

[0042] 2. Fractionation of enriched cross-linked peptides and determination of cross-linked sites

[0043] The cross-linked peptides released in step (1) were desalted, lyophilized, fractionated by cation exchange separation, desalted, lyophilized and redissolved in 0.1% formic acid solution for mass spectrometry analysis.

[0044] Identification result

[0045]

[0046]

[0047]

Embodiment 3

[0049] 1. Cross-linking reaction of protein samples

[0050] Dissolve 10 μg of rabbit creatine kinase protein sample (CK) in 50 mM phosphate-buffered saline (PBS) with a pH of 7.4, the final protein concentration is 1 mg / mL, and prepare DST with a concentration of 25 mM in 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.

[0051] 2. Removal of excess cross-linking agent

[0052] 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.

[0053] 3. Dissolution, denaturation and reduction of protein samples

[0054] 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.

[0055] 4. Alkylation and enzymatic hydrolysis of...

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Abstract

The invention relates to a cross-linked peptide enrichment method and its application in protein interaction research. The intracellular protein complexes were cross-linked and enzymatically digested using a cross-linking agent with reactive groups on both sides and adjacent dihydroxyl groups on the linker, followed by selective enrichment of the cross-linked peptides using a boron affinity material. collection and efficient release. The method has the advantages of simple operation, high efficiency, high throughput and high reliability, and it is applied to the analysis of protein interaction.

Description

technical field [0001] The invention relates to a cross-linked peptide enrichment method and its application in protein interaction research. Background technique [0002] Cross-linking mass spectrometry is a new technology developed in the past ten years. It uses a chemical cross-linking agent to covalently link two amino acids that are close enough in the cell space to react with the cross-linking agent. The proteomics of mass spectrometry analyzes the cross-linked products, so as to realize the analysis of the spatial structure of proteins and the interaction mode between proteins (Sinz, A., Expert Rev. Proteomics., 2014, 11(6): 733-743 .). [0003] However, the use of traditional cross-linked mass spectrometry requires high mass spectrometry identification. Since the actual cross-linking efficiency of each site is often far below 100%, the abundance of cross-linked peptides is low, the number of cross-linked spectra is small, and the signal is poor, which brings diffic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/40G01N27/62
Inventor 张丽华方菲赵群安雨馨杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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