The development of BioID: A tool for proximity proteomics in living cells

Mapping and identification of proteins and protein complexes in cells is of fundamental importance when studying protein functions and biological mechanisms. Conventional methods, such as affinity purification coupled with mass spectrometry (AP-MS) and yeast two-hybrid screening, have commonly been employed to study protein-protein interactions. The limitations associated with these methods have led to the development of proximity-dependent labelling (PDL) coupled with mass spectrometry, a molecular technique developed to map proteins in the immediate vicinity of a protein of interest in living cells. Proximity-dependent biotin identification (BioID) is a PDL technique that uses a modified, mutated biotin protein ligase to catalyse labelling of proximal proteins. Recently, a range of biotin protein ligases (BPLs) have been developed to enhance enzymatic efficiency, versatility, and labelling speed. This review article describes the molecular basis of BioID, details the development of various mutant BPLs, and provides a comparative overview of the advantages and disadvantages of each BPL variant. We further address the experimental considerations that determine whether a proximity labelling dataset is interpretable, including construct design and expression level, the selection of matched controls, labelling conditions, the distinction between protein-level and site-level enrichment, and the quantitative and statistical treatment of the resulting data. Finally, we consider how BioID complements rather than replaces affinity purification, imaging and functional assays, and how it is combined with orthogonal validation, time-resolved designs and other omics measurements.