Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs Dev Biol
Impact Factor: 4.711

Proximity‐dependent labeling methods for proteomic profiling in living cells

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

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 with biotin. The biotinylated 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 describe and compare current methods of proximity labeling as well as their applications. 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.

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 biotinylated endogenous proteins are isolated using steptavidin beads. Small peptides of enriched proteins are generated by trypsin digestion and subsequently ionized for mass spectrometry analysis. The mass‐to‐charge (m/z) ratio of each peptide is then used to identify peptide sequence usually through computational comparison against established databases.
[ Normal View | Magnified View ]
Proximity labeling methods. BioID, a mutant form of the biotin ligase BirA, can convert biotin into radicals that can covalently tag neighboring proteins on lysine residues. 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. In addition, fluorescein‐aryl azide or biotin‐aryl azide have been used for HRP‐mediated proximity labeling (not shown in the figure). HRP is inactive in a reducing environment, such as the cytosol, but functions extracellularly. APEX, engineered ascorbate peroxidase; BioID, proximity‐dependent biotin identification; HRP, horseradish peroxidase; SP, spatially restricted protein.
[ Normal View | Magnified View ]

Browse by Topic

Technologies > Analysis of Proteins

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts

Twitter: molecular Follow us on Twitter

    Protein Science 2017 Best Paper Award Winners: Congratulations to Zach Schaefer. Read his winning paper for free… https://t.co/12clsdAVor
    Protein Science 2017 Best Paper Award Winners: Congratulations to Charlotte Miton. Read her winning paper for free… https://t.co/f35tR1K1lX