Sumoylation peptide fragment enrichment method based on desumoylation enzyme and SAX

A peptide and pre-enrichment technology, which is applied in chemical instruments and methods, peptide preparation methods, peptides, etc., can solve problems such as inability to accurately reflect the real modification state of proteins, and achieve improved identification coverage and high enrichment selectivity , reduce the effect of identifying false positives

Active Publication Date: 2021-06-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Sumoylation peptide fragment enrichment method based on desumoylation enzyme and SAX
  • Sumoylation peptide fragment enrichment method based on desumoylation enzyme and SAX
  • Sumoylation peptide fragment enrichment method based on desumoylation enzyme and SAX

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Embodiment 1

[0037] Such as figure 1 As shown, first digest the basic site of the protein, and then block the N-terminal of the peptide and the free amino group of the side chain at the peptide level. The retention in anion-exchange chromatography is stronger than that of non-SUMOylated peptides, so anion-exchange chromatography is used to pre-enrich the SUMOylated peptides with strong retention, and then the collected SUMOylated peptides are deSUMOlated. After de-SUMOylation, the retention of de-SUMOylated peptides in anion-exchange chromatography becomes weaker, so that the enrichment of SUMOylated peptides is achieved in the second anion-exchange chromatography.

[0038] The SUMO1 standard peptide with the sequence ELGMEEEDVIEVYQEQTGG(19)-LLVHMGLLKSEDKVK(9) was used as the sample, dissolved in 50mM ammonium bicarbonate, and digested with deSUMOlase SENP1, wherein the amount of enzyme was 1 / 20 of the sample mass, and the temperature was After enzymatic hydrolysis for 12 hours at 37°C, p...

Embodiment 2

[0040]Redissolve 10 μg of SUMO1 standard peptides in phase A, load the sample onto a tertiary amine-based ion exchange column (4.6mm i.d×5cm) for isocratic elution, elution gradient (V / V): 0-10min, 20% B; 10-20min, 80% B, flow rate is 1.0mL / min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. Such as image 3 As shown, after the unretained fraction 10 minutes before elution, the fraction containing the SUMO1-labeled peptide 10 minutes later was collected, desalted, and lyophilized. It was redissolved in 0.1% formic acid and analyzed by mass spectrometry. It can be seen that the SUMO1 standard peptide was effectively retained and enriched in anion exchange chromatography.

Embodiment 3

[0042] 10 μg of SUMO1ylated standard peptides were digested with deSUMOlase, loaded onto a tertiary amine-based ion exchange column (4.6mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 20% B; 10-20min, 80% B, flow rate is 1.0mL / min. Phase A: 20% acetonitrile + 1 mM pH 8 Tris buffer; Phase B: 20% acetonitrile + 1 mM pH 8 Tris buffer + 500 mM sodium chloride. Such as Figure 4 As indicated, the non-retained fractions in the first 10 minutes and the retained fractions after 10 minutes were collected, desalted, and freeze-dried. Redissolved in 0.1% formic acid and analyzed by mass spectrometry, the de-SUMOylated peptide LLVHMGLLKSEDKVK after being digested by de-SUMOlase does not retain the outflow in anion exchange, the original SUMO1-ylated standard peptide and the peptide obtained after digestion ELGMEEEDVIEVYQEQTGG was still retained in anion exchange, indicating that the effective enrichment and identification analysis of SUMOylated peptides was successfully ac...

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Abstract

The invention relates to a sumoylation peptide fragment enrichment method based on a desumoylation enzyme and SAX. Firstly, enzymolysis is performed on alkaline sites of a protein sample into polypeptides, and free amino groups at the N tail end and side chain of the peptides are horizontally sealed at the peptide fragments; then, the SAX is adopted for pre-enriching to retain stronger sumoylation peptide fragments in polypeptides; then, the collected sumoylation peptide fragments are subjected to desumoylation by a desumoylation enzyme, and the retention of the peptide fragments after the desumoylation becomes weak in the SAX; the sample passes through the SAX again, and unretained fractions are collected; and desumoylation modification substrate peptide fragments are separated from other interference peptide fragments to realize secondary enrichment of the sumoylation modification peptide fragments. The method has the advantages that the selectivity is high, the enrichment efficiency is high, various types of sumoylation modification peptide fragments can be enriched at the same time, and the identification coverage of the sumoylation modification sites is improved.

Description

technical field [0001] The present invention relates to a method for enriching SUMOylated peptides, that is, a method for enriching SUMOylated peptides based on de-SUMOlase and anion exchange chromatography (SAX), in order to achieve efficient and efficient sumoylated peptides in complex protein samples. Highly selective enrichment. 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. [0003] SUMO plays an important regulatory role in multiple cellular physiological activities, such as maintaining genome stability, regulating cell cycle, regulating cell differentiation and transcri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/18
CPCC12P21/06C07K1/18
Inventor 张丽华李洋单亦初杨开广梁振张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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