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Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

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Abstract Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling‐based methods coupled with mass spectrometry (MS) offer a high‐throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins. The tagged endogenous proteins can then be isolated for further analysis by MS. To analyze protein–protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we provide an updated description and comparison of proximity labeling methods, as well as their applications and improvements. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions. This article is categorized under: Technologies > Analysis of Proteins
Proximity labeling for proteomic profiling. To achieve regional protein labeling, the enzymes are usually fused with a targeting signal peptide or a spatially restricted protein (SP). The enzymes can also be fused with any protein of interest for protein interactome studies. After performing proximity labeling in living cells, the cells are lysed and the tagged endogenous proteins are isolated using steptavidin beads. Small peptides of enriched proteins are generated by trypsin digestion and subsequently ionized for tandem mass spectrometry (MS/MS) analysis. The mass‐to‐charge (m/z) ratio of each peptide and their fragment ions is then used to identify peptide sequence through computational comparison against established databases
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Directed evolution of proximity labeling components. Proximity labeling enzymes have been modified from their wild‐type counterparts by selecting for mutants with promiscuous activity. Directed evolution has been used to isolate enzymes with increased activity, increased stability, smaller molecular weight, and that are split into inactive fragments that reconstitute activity when combined. Smaller Pup substrates have also been identified
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Proximity labeling methods. HRP and APEX are peroxidases that, when activated by H2O2, are able to turn biotin‐phenol substrates into highly reactive radicals that covalently tag neighboring proteins on electron‐rich amino acids. HRP is inactive in a reducing environment, such as the cytosol, but functions extracellularly. BioID, a mutant form of the biotin ligase BirA, can convert biotin into radicals that can covalently tag neighboring proteins on lysine residues. PafA is a ligase that can covalently tag neighboring proteins with the small protein Pup onto lysine residues. APEX, engineered ascorbate peroxidase; BioID, proximity‐dependent biotin identification; HRP, horseradish peroxidase
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