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A single-end cross-linked peptide removal method and its application to the analysis of cross-link sites in protein complexes

A protein and protein technology, applied in the analysis of materials, material analysis by electromagnetic means, biological testing, etc., can solve the problem of low abundance of cross-linked peptides, complex cross-linked samples, and the inability to achieve selective enrichment of cross-linked peptides and other problems, to achieve the effect of combining fast and efficient, high enrichment selectivity, and improved identification sensitivity

Active Publication Date: 2022-05-06
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

[0006] In order to overcome the problems of complex cross-linked samples, low abundance of cross-linked peptides, and inability to achieve selective enrichment of cross-linked peptides by conventional chemical cross-linking methods, the present invention provides a split-type cross-linking reagent. After the reaction is completed, the unreacted functional groups are blocked with a blocking reagent, and then the cross-linked peptides are selectively enriched and released efficiently by using highly specific enrichment materials

Method used

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  • A single-end cross-linked peptide removal method and its application to the analysis of cross-link sites in protein complexes
  • A single-end cross-linked peptide removal method and its application to the analysis of cross-link sites in protein complexes
  • A single-end cross-linked peptide removal method and its application to the analysis of cross-link sites in protein complexes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Cross-linking reaction of protein samples

[0026] 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) Propargyl nitroheptyl disuccinimidyl ester (BSPNO) was prepared at a concentration of 25 mM, and 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.

[0027] 2. Blocking of unreacted NHS groups

[0028] Add amino-PEG at a final concentration of 10 mM 3 -Biotin, react at room temperature for 2h.

[0029] 3. Click chemistry

[0030] Add 20mM CuSO to the sample 4 , 160mM THPTA and 500mM Vc to make the final concentrations of 100μM, 800μM and 2.5mM respectively, add 20mM Diazo Biotin-Azide to make the final concentration of 200μM, and react at 60°C for 2h.

[0031] 4. Protein precipitation

[0032] Add four times ...

Embodiment 2

[0051] 1. Cross-linking reaction of protein samples

[0052] 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) Azidonitroheptyl disuccinimide ester was prepared at a concentration of 25 mM, the cross-linking agent was added to the BSA solution so that the final concentration was 1 mM, and the reaction was carried out at room temperature for 1 h.

[0053] 2. Blocking of unreacted NHS groups

[0054] Add amino-PEG at a final concentration of 10 mM 3 -Biotin, react at room temperature for 2h.

[0055] 3. Click chemistry

[0056] Add 20mM CuSO to the sample 4 , 160mM THPTA and 500mM Vc to make the final concentrations of 100μM, 800μM and 2.5mM respectively, add 20mM Alkyne Biotin-Azide with a final concentration of 200μM, and react at 60°C for 2h.

[0057] 4. Protein precipitation

[0058] Add eight ti...

Embodiment 3

[0079] 1. Cross-linking reaction of protein samples

[0080] 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 use dimethylformamide (DMF) to prepare 25 mM propargyl nitric acid Heptyl disuccinimidyl ester (BSPNO), 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.

[0081] 2. Blocking of unreacted NHS groups

[0082] Add amino-PEG11-biotin at a final concentration of 10 mM, and react at room temperature for 2 h.

[0083] 3. Click chemistry

[0084] Add 20mM CuSO4, 160mM BTTAA and 500mM Vc to the sample to make the final concentrations of 100μM, 800μM and 2.5mM respectively, add 20mM Diazo Biotin-Azide with a final concentration of 200μM, and react at 37°C for 1h.

[0085] 4. Protein precipitation

[0086] Add four times the volume of pre-cooled acetone to the sample, and p...

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Abstract

The invention relates to a method for removing single-end reaction peptides in protein complex analysis based on chemical cross-linking mass spectrometry and its application in the analysis of protein complex cross-linking sites. The split-type cleavable and enrichable cross-linking reagent is used, and the same blocking reagent as the cross-linked peptide enrichment group can be used to realize the enrichment and single-end reaction of cross-linked peptides at the same time without increasing the experimental steps Peptide removal, thereby increasing the number and confidence of identification of cross-linked peptides at both ends. 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 method for reducing the abundance of single-end cross-linked peptides, thereby reducing the complexity of cross-linked samples, thereby improving the identification of cross-linked peptides and its application in the analysis of protein complex cross-linking sites. 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N27/62G01N1/28
CPCG01N33/6848G01N27/62G01N1/28
Inventor 张丽华安雨馨赵群高航张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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