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A method for enrichment of sumoylated peptides based on de-sumoylase and sax

A peptide and enzyme technology, applied in the field of enrichment of SUMOylated peptides, can solve problems such as the 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: 2022-08-09
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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  • A method for enrichment of sumoylated peptides based on de-sumoylase and sax
  • A method for enrichment of sumoylated peptides based on de-sumoylase and sax
  • A method for enrichment of sumoylated peptides based on de-sumoylase and sax

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Experimental program
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Effect test

Embodiment 1

[0037] like figure 1 As shown in the figure, the basic site of the protein is first digested, and then the N-terminus of the peptide and the free amino group of the side chain are blocked 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 used to remove SUMOylase After de-SUMOylation, the retention of the peptides after de-SUMOylation in anion exchange chromatography is weakened, so that the enrichment of SUMOylated peptides is achieved in the second anion exchange chromatography.

[0038] Take the SUMO1 standard peptide with the sequence ELGMEEEDVIEVYQEQTGG(19)-LLVHMGLLKSEDKVK(9) as the sample, dissolve it in 50mM ammonium bicarbonate, and digest it with the de-SUMOylase SENP1, where the amount of enzyme is 1 / 20 of the sample mass, and the temperature is After e...

Embodiment 2

[0040]Redissolve 10 μg of SUMO1 standard peptide in phase A, load the sample into a tertiary amine ion exchange column (4.6 mm i.d × 5 cm) for isocratic elution, elution gradient (V / V): 0-10 min, 20% B; 10-20 min, 80% B, flow rate 1.0 mL / 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. like image 3 As shown, after the unretained fractions 10 min before elution, the fractions containing the SUMO1 target peptide after 10 min were collected, desalted, and lyophilized. After redissolving in 0.1% formic acid, mass spectrometry analysis showed that the SUMO1 standard peptide was effectively retained and enriched in anion exchange chromatography.

Embodiment 3

[0042] 10μg of SUMO1 standard peptide fragment was digested with de-SUMOylase and loaded onto a tertiary amine ion exchange column (4.6mm i.d×5cm) for isocratic elution, elution gradient: 0-10min, 20%B; 10-20 min, 80% B, flow rate 1.0 mL / 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. like Figure 4 As shown, the unretained fractions in the first 10 min and the retained fractions after 10 min were collected, demineralized, and lyophilized. Redissolved in 0.1% formic acid, and subjected to mass spectrometry analysis, the de-SUMOylated peptide LLVHMGLLKSEDKVK after being digested by de-SUMOylase did not retain efflux in anion exchange, the original SUMO1 standard peptide and the peptide obtained after digestion ELGMEEEDVIEVYQEQTGG still remained in the anion exchange, indicating that the effective enrichment and identification analysis of SUMOylated peptides was successfully achieved.

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Abstract

The invention relates to a method for enriching SUMO modified peptide segments based on de-SUMOylase and anion exchange chromatography. Firstly, the alkaline site of a protein sample is enzymatically decomposed into polypeptides, and the N-terminal and The free amino group of the side chain is then used to pre-enrich the peptide by anion exchange chromatography to retain strong SUMOylated peptides, and then the collected SUMOylated peptides are de-SUMOylated with de-SUMOylated enzyme, and the de-SUMOylated peptides are de-SUMOylated. The retention of peptides in anion exchange chromatography will be weakened. The sample is again subjected to anion exchange chromatography to collect the unretained fractions. By separating the de-SUMO modified substrate peptides from other interfering peptides, SUMO Secondary enrichment of modified peptides. The invention has the advantages of high selectivity, high enrichment efficiency, simultaneous enrichment of multiple types of SUMO modified peptides, and improved identification coverage of SUMO modified sites.

Description

technical field [0001] The present invention relates to a method for enriching SUMOylated peptides, namely a method for enriching SUMOylated peptides based on de-SUMOylase and anion exchange chromatography (SAX), so as to achieve high efficiency and high efficiency of 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 first modification that is found to be linked to proteins, and it plays an important role in protein degradation. In the past ten years, scientists have discovered some ubiquitin-like proteins, among which small ubiquitin-like modifiers (SUMO) are the most attention-seeking class. [0003] 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(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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