A method for the enrichment of sumoylated peptides based on anion-exchange chromatographic columns

A technology of exchange chromatography and anion, which is applied in the field of enrichment of SUMOylated peptides, can solve problems such as inability to accurately reflect the real modification state of proteins, and achieve the effects of improving identification coverage, high enrichment selectivity, and promoting efficient removal

Active Publication Date: 2022-05-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

This type of method helps to improve the identification efficiency of SUMO modification, but it is only applicable to biological samples (such as cells) that can be changed in genes, and it is powerless for samples such as tissues and blood; in addition, changes to the SUMO sequence may affect the properties of SUMO and function, so cannot accurately reflect the true modification state of the protein in the sample
Several recent articles have been reported to successfully identify endogenous SUMOylated peptides in human cells and mouse tissues (Nature Communication 2018, 9, 2456; Nature Communication 2017, 8, 1171; Molecular&Cellular Proteomics 2017 , 16, 717-727), but still rely on expensive antibodies for affinity enrichment of SUMO-modified peptides, and can only be enriched for a class of SUMO-modified peptides

Method used

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  • A method for the enrichment of sumoylated peptides based on anion-exchange chromatographic columns
  • A method for the enrichment of sumoylated peptides based on anion-exchange chromatographic columns
  • A method for the enrichment of sumoylated peptides based on anion-exchange chromatographic columns

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Embodiment 1

[0029] Such as figure 1 As shown, the basic site of the protein is first digested, and the SUMOylated peptide after digestion has multiple acidic amino acids, and the retention in anion exchange chromatography in an alkaline environment is stronger than that of the non-SUMOylated peptide. Anion-exchange chromatography was used to elute the weakly retained enzyme cleavage products first, and finally the peptide fractions with strong retention were collected, which were the enriched SUMOylated peptides.

[0030] Take HeLa cells as samples, dissolve 100 μg of extracted protein in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), add 10 μl of 100 mM dithiothreitol, denature at 56°C for 1 hour, then add 10 μl of 300 mM iodoacetamide to react After 0.5h, add another 30μl of 100mM dithiothreitol, incubate for 10min, digest with trypsin, wherein the amount of enzyme is 1 / 10 of the sample mass, the temperature is 37℃, after 60min of enzymolysis, desalt, freeze-dry , redis...

Embodiment 2

[0032] Take HeLa cells as samples, dissolve 100 μg of extracted protein in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), add 10 μl of 100 mM dithiothreitol, denature at 56°C for 1 hour, then add 10 μl of 300 mM iodoacetamide to react After 0.5h, another 30μl of 100mM dithiothreitol was added, and after incubation for 10min, it was digested with Lys-C, wherein the enzyme dosage was 1 / 10 of the sample mass, and the temperature was 37°C. Dried, redissolved in 0.1% formic acid, and analyzed by mass spectrometry, the lysine at the carboxy-terminal of the peptide was cleaved efficiently and selectively.

Embodiment 3

[0034] Take HeLa cells as samples, dissolve 100 μg of extracted protein in 100 μl of 8M urea in 50 mM ammonium bicarbonate buffer (pH 8), add 10 μl of 100 mM dithiothreitol, denature at 56°C for 1 hour, then add 10 μl of 300 mM iodoacetamide to react After 0.5h, add another 30μl 100mM dithiothreitol, incubate for 10min, and use Arg-C to digest, wherein the amount of enzyme is 1 / 10 of the sample mass, and the temperature is 37℃. After 60min of enzymatic hydrolysis, desalt, freeze Dried, redissolved in 0.1% formic acid, and analyzed by mass spectrometry, the arginine at the carboxy-terminal of the peptide was cleaved efficiently and selectively.

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Abstract

The present invention relates to a method for enriching SUMOized peptides based on an anion exchange chromatography column, comprising: enzymatic digestion of protein alkaline sites, removing weakly retained enzymatic peptides through an anion exchange chromatography column in an alkaline environment, collecting Stronger SUMOylated peptide eluate is retained. First, digest the basic site of the protein. The SUMOylated peptide after digestion has multiple acidic amino acids, and its retention in the anion-exchange column is stronger than that of the non-SUMOylated peptide in an alkaline environment. The anion-exchange chromatography column first elutes the digested products with weaker retention, and finally collects the peptide fraction with stronger retention, which is the enriched SUMOylated peptide. The invention has the advantages of simple and convenient operation steps, high selectivity, high enrichment efficiency, simultaneous enrichment of multiple types of SUMOylated peptides, and improved identification coverage of SUMOylated modification sites.

Description

technical field [0001] The invention relates to a method for enriching SUMOylated peptides, that is, a method for enriching SUMOylated peptides based on anion-exchange chromatographic columns, so as to realize efficient and highly selective enrichment of SUMOylated peptides in complex protein samples. Background technique [0002] Ubiquitination is one of the common post-translational modifications in organisms, and it is also the earliest discovered modification method connected to proteins, which plays an important role in protein degradation. In the past ten years, scientists have successively discovered some ubiquitin-like proteins, among which small ubiquitin-like modifiers (small ubiquitin-like modifiers, SUMO) are the most concerned one. SUMO plays an important regulatory role in multiple cellular physiological activities, such as maintaining genome stability, regulating cell cycle, regulating cell differentiation and transcription factor activity, and participating i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/08G01N30/14
CPCG01N30/08G01N30/14
Inventor 张丽华李洋孙明伟单亦初杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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