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Non-antibody-dependent protein methylation modification enrichment analysis method

An analytical method and methylation technology, applied in analytical materials, material separation, measurement devices, etc., can solve the problems of difficult to achieve deep coverage analysis of protein methylation modification, low enrichment efficiency of methylated modified peptides, etc. Achieve high-throughput analysis, improve selectivity, and reduce interference

Inactive Publication Date: 2020-05-29
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

Since this method uses a strong cation exchange column to enrich methylated modified peptides, it is easily interfered by positively charged amino acids under alkaline conditions, and the enrichment efficiency of methylated modified peptides is low. Difficult to achieve deep coverage analysis of protein methylation modifications

Method used

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  • Non-antibody-dependent protein methylation modification enrichment analysis method

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Embodiment 1

[0025] like figure 1As shown, after the protein is reductively alkylated, the protein is digested with Lys-C / Trypsin / Arg-C three enzymes, and then the peptides obtained by enzyme digestion are immobilized with amino active materials, and further used The enzyme Lysarginase or Trypsin-N with enzyme-cutting methylated modified lysine and arginine can digest the immobilized peptide, and use affinity chromatography or molecular sieve to separate the digested methylated peptide from the material , so as to realize the enrichment of methylated modified peptides, and analyze the enriched methylated modified samples by high performance liquid phase reverse chromatography-ultra-high resolution mass spectrometry (LC-MS / MS).

[0026] Using cervical cancer Hela cells as samples, 8M urea was used as the lysate for protein extraction, the extracted protein was quantified by BCA, and 1 mg of protein was taken for analysis of methylation modification. Add dithiothreitol to a final concentrat...

Embodiment 2

[0028] Using cervical cancer Hela cells as samples, protein was extracted using 6M guanidine hydrochloride as lysate, the extracted protein was quantified by BCA, and 1 mg of protein was taken for analysis of methylation modification. Add dithiothreitol to a final concentration of 10mM, after denaturation and reduction at 56°C for 30min, add iodoacetamide to a final concentration of 30mM, react at room temperature in the dark for 30min, then add dithiothreitol to the reaction system to a final concentration of 30mM, incubate at room temperature for 30min to terminate excess iodoacetamide. Add 10 μg of Lys-C, adjust the pH to 7.4, digest at 37°C for 4 hours, then dilute guanidine hydrochloride to 0.8M with 50 mM ammonium bicarbonate solution, add 20 μg of Trypsin, and digest at 37°C for 12 hours. Centrifuge at 15,000 rcf for 20 minutes to obtain enzyme-cleaved peptides, and immobilize the centrifuged peptide samples on a C18 reversed-phase trapping column (4.6mm i.d.×1cm) for d...

Embodiment 3

[0030] Take cervical cancer Hela cells as samples, use 6M guanidine hydrochloride as the lysate to extract protein, after the extracted protein is reductively alkylated, add 10 μg of Lys-C, adjust the pH to 7.4, and digest at 37 degrees for 4 hours , and then use 50 mM ammonium bicarbonate solution to dilute urea to 0.8 M, add 40 μg of Trypsin, and digest at 37° C. for 6 h. Centrifuge at 15,000 rcf for 20 minutes to obtain enzyme-cleaved peptides, and immobilize the centrifuged peptide samples on a C18 reversed-phase trapping column (4.6mm i.d.×1cm) for desalting, and use 0.1% trifluoroacetic acid / 80% acetonitrile for elution, After freeze drying. Then the dried peptide was dissolved in 20mM HEPES containing 5mM calcium chloride, 5mM dithiothreitol, and 2mM EDTA, and the pH was adjusted to 7.4, and 10μg of Arg-C was added to adjust the pH to 7.4 , Digest overnight at 37 degrees. Use C18 to desalt and dry the sample, add 5mg of HPG-ALD and 20mM sodium cyanoborocyanide to the ...

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Abstract

The invention relates to a novel non-antibody-dependent protein methylation modification enrichment analysis method and application. The method comprises the steps of Lys-C / Trypsin / Arg-C continuous enzyme digestion, immobilization of N-segment amino groups of an enzyme digested peptide segment, selective release of a methylation modified peptide segment, LC-MS / MS analysis and data analysis of methylation modification site information. Firstly, Lys-C / Trypsin / Arg-C multiple enzyme digestion is performed on a sample containing protein methylation modification, then the peptide fragments obtainedthrough continuous enzyme digestion react with an amino reaction active material, immobilization of the peptide fragments is achieved, and finally specific enzyme digestion is conducted on methylation-modified lysine and arginine N-terminal peptide bonds contained in the immobilized peptide fragments through specific enzyme. The method has the advantages that simultaneous enrichment of methylationmodification of various types of low-abundance proteins is realized, including lysine dimethylation modification, lysine trimethylation modification, arginine monomethylation modification and arginine dimethylation modification.

Description

technical field [0001] The invention relates to a new method and application of non-antibody-dependent protein methylation modification enrichment analysis, which realizes the simultaneous analysis of various types of protein methylation modifications. Background technique [0002] Protein methylation modification is one of the post-translational modifications that exist widely and have important biological functions. For example, histone methylation modification participates in various life activities such as gene transcription, DNA damage repair, and signal transduction. Abnormal regulation of protein methylation modification is closely related to various diseases. For example, abnormal regulation of protein methylation modification is closely related to breast cancer, lung cancer, lymphoma, AIDS and cardiovascular diseases. In addition, the dynamic changes of protein methylation play an important role in regulating life activities. For example, Jarid2 protein 116 lysine m...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/08
CPCG01N30/08G01N30/88G01N2030/8831
Inventor 张丽华孙明伟梁振单亦初赵宝锋杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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