An enzymatic cascade enables sensitive and specific proximity labeling proteomics in challenging biological systems

Abstract

Abstract Ascorbate peroxidase (APEX) is a proximity labeling enzyme used for subcellular proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. To minimize toxicity and reduce non-specific background labeling we establish a more versatile in situ APEX activation (iAPEX) workflow by combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide. Using iAPEX, we profile the proteomes of a cellular microdomain, the primary cilium, in cell lines not readily accessible to conventional APEX labeling and identify unknown ciliary proteins. Our study validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. Furthermore, iAPEX proximity labeling is applicable to a range of cellular compartments including mitochondria and lipid droplets and can be employed in Xenopus laevis , which provides a proof-of-concept for future in vivo applications.