Proteomic proximity labeling to reveal interactions between biomolecules

Abstract

There are currently several methods that address proteomic proximity labeling, and that depend on the biological question asked and localization in the cell. These include BioID, APEX, EMARS, and SPPLAT. Here we describe SPPLAT, a method that can identify members of protein microenvironments localized to the plasma membrane, as well as proteins that interact with each other in endocytic pathways. The SPPLAT protocol is particularly useful as a discovery-based approach, to identify novel molecular neighbors of a predetermined plasma membrane protein target. It allows a quick survey of the target proteins’ environment without the need for genetic manipulation. By using various readily available biotin-reactive reagents, together with suitable antibodies, drugs, or toxins directed to a protein target, the user can vary the amount of labeling and can decide to keep or cleave the covalent tag for downstream applications. Proteins and other macromolecules that are specifically biotin tagged can easily be purified and then identified my mass spectrometry, thus allowing one to build a map of cell-surface protein microenvironments that are often the target for therapeutics.