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Screening method and application of uridine monophosphate modified protein in mitochondria

A technology of uridine monophosphate and screening method, which is applied in the field of screening of uridine monophosphate-modified proteins in mitochondria, and can solve problems such as limiting the research progress of SelO protein and mitochondrial signal transduction

Inactive Publication Date: 2020-09-22
INST OF BASIC MEDICINE OF SAMS
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  • Claims
  • Application Information

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Problems solved by technology

However, in eukaryotes, no substrates for SelO-catalyzed uridine modification of monophosphate have been found, which greatly limits the research progress of SelO protein and mitochondrial signal transduction.

Method used

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  • Screening method and application of uridine monophosphate modified protein in mitochondria
  • Screening method and application of uridine monophosphate modified protein in mitochondria
  • Screening method and application of uridine monophosphate modified protein in mitochondria

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Embodiment Construction

[0029] The present invention will be further described according to the following examples, and the mode of the present invention includes but not limited to the following examples.

[0030] The present invention comprises the following steps:

[0031] A The total mitochondrial protein is obtained by extracting and lysing the total mitochondrial protein after cell culture;

[0032] B. After the desalting column treatment of the total mitochondrial protein, the Biotn-UMP labeled protein is obtained after the UMP modification reaction;

[0033] C after screening the Biotn-UMP tagged protein;

[0034] The Biotn-UMP tagged protein in D is identified and screened by chromatography to determine the modified protein and the modified site.

[0035] The mass spectrometry identification includes that the eluate of the Biotn-UMP labeled protein is subjected to 12% SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining or silver staining, and the gel is cut for mass spectrom...

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Abstract

The invention discloses a screening method and an application of uridine monophosphate modified protein in mitochondria. The method is characterized in that biotin-labeled UTP (Biotin-16-UTP) is usedas a raw material, under the action of uridine monophosphate transferase SelO, total mitochondrial protein is subjected to UMP modification reaction, and the protein modified by UMP further has a biotin label, subsequently, the modified protein is screened out by using a biotin-streptavidin Pull-down technology, and finally, the modified protein and a modification site are determined by mass spectrometry. The method is advantaged in that a simple and feasible experimental method is developed, proteins which can be modified by UMP are screened from mitochondrial total proteins, and an importantresearch means is provided for signal transduction mediated by SelO proteins and post-translational modification in mitochondria.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a screening method and application of monophosphate urylation modified protein in mitochondria. Background technique [0002] Mitochondria are organelles unique to eukaryotes, which play an important role in cellular energy metabolism, signal transduction, and cell survival regulation. Many human diseases are related to mitochondrial abnormalities. At present, the signal transduction process in mitochondria has not been fully grasped by humans. [0003] SelO (Seleno O, selenoprotein O) is a selenoprotein that has a mitochondrial localization sequence and exists in mitochondria after synthesis. Under selenium-deficient conditions, the synthesis of SelO proteins takes precedence over other selenoproteins. The expression of SelO in tumor tissue was significantly lower than that in normal tissue. Moreover, the high expression of SelO is also an evaluation index for better prognosis of v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/02
CPCG01N33/6839G01N33/6848G01N30/02
Inventor 李冰清岳盈盈宋楠楠杨银龙李翠玲王玮玮贾海红
Owner INST OF BASIC MEDICINE OF SAMS
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