Compartmentation is one of the characteristics of eukaryotic cells, and different biochemical processes can be divided by spatial structure. Microscopy and mass spectrometry analysis can be used to analyze the proteome of different organelles, but it is still difficult to directly use these two methods to identify the interactomics in many intracellular compartments.
In order to identify interactomes in different compartments in human cells, on June 2, 2021, the Anne-Claude Gingras research group of the Lunenfeld-Tanenbaum Institute in Canada issued a paper entitled A proximity-dependent biotinylation map of a human cell, A map of human cells was established by means of biotinylation of adjacent markers.
The biotin-dependent proximity labeling method can be used to describe the intracellular environment occupied by the proteins in the cell. For example, BioID technology uses the biotin ligase BirA* (* means mutant), which contains a R118G site mutation, which can be used as a "bait" to fuse with the target protein and expressed in cultured cells or in a multicellular system. in. The biotinylated protein can then be captured by streptavidin, and the interacting protein can be analyzed by mass spectrometry identification. Since the average diameter of the globular protein is 5-10nm, the labeling radius of this technology can directly biotin label the interacting protein. At present, BioID technology has been successfully used to define the composition of many different protein complexes, the identification of membrane binding and the spatial organization of membraneless organelles.
In order to further identify the interacting proteome in human living cells, the authors used BioID technology to label 234 intracellular proteins in 32 different cell components as "bait" to catch interacting proteins to establish The structure of protein organization in living human cells. Each bait protein will be labeled with BirA*, and then a stable expression line of HEK cells will be established. By comparing the identified interacting proteins with the negative control, the authors evaluated the reliability of the interaction factors caught. A total of 192 candidate protein markers passed the quality assessment, and a total of 35,902 interactions were identified. Action factor.
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