Proximity-Dependent Biotinylation Approaches to Explore the Dynamic Compartmentalized Proteome

Abstract

In recent years, proximity-dependent biotinylation approaches, including BioID, APEX, and their derivatives, have been widely used to define the compositions of organelles and other structures in cultured cells and model organisms. The associations between specific proteins and given compartments are regulated by several post-translational modifications (PTMs); however, these effects have not been systematically investigated using proximity proteomics. Here, we discuss the progress made in this field and how proximity-dependent biotinylation strategies could elucidate the contributions of PTMs, such as phosphorylation, to the compartmentalization of proteins.

Keywords: APEX; BioID; cellular organization; mass spectrometry; phosphorylation; post-translational modification; proximity-dependent biotinylation.

FIGURE 1

PDB strategies to study the contributions of phosphorylation to proteome compartmentalization (A) Combining PDB with phosphopeptide enrichment. A biotinylating enzyme (red rectangle, biotin is represented by orange circles) is targeted to a specific cellular structure to label its spatial subproteome. Streptavidin affinity purification is followed by TiO₂ enrichment to identify local phosphosites. This concept could be applied to other PTM enrichment strategies as well. (B) Protein engineering strategies, such as split-PDB systems, can be used to investigate the consequences of spatially resolved PTMs such as phosphorylation. A reader domain for a specific type of phosphorylation is fused to one half of the PDB enzyme and the substrate of interest (localized to a specific subcompartment) is fused to the other half. The impacts of phosphorylation of the protein of interest can then be determined via PDB-MS. (C) Combining PDB tools (red rectangle and orange circles) with orthogonal approaches (curved arrows). PDB is achieved in a specific cellular structure and PTM-generating enzymes are modulated via orthogonal strategies (e.g., optogenetically or chemically). Impacts on the spatial proteome are determined using streptavidin affinity purification and MS. Figure generated with BioRender.com.