Best Practices for Biotin Ligase-based Proximity Labeling Proteomics in Plant Systems.

Proximity labeling (PL) proteomics, primarily powered by engineered biotin ligases such as BioID and TurboID, has emerged as a transformative approach for mapping protein association networks and subcellular proteomes in living cells. By covalently biotinylating proteins within a nanometer-scale radius of a bait protein, PL captures transient, weak, and spatially restricted associations that often escape conventional affinity-based methods. However, applying PL in plants introduces distinctive challenges, including rigid cell walls that can limit biotin penetration, tissue-specific variation in substrate accessibility, enzyme temperature sensitivity, and the difficulty of removing excess free biotin after labeling. Here, we outline best practices for designing and implementing biotin ligase-based PL experiments in plant systems, drawing on experience across multiple species. We discuss key considerations, including enzyme selection, expression system design, fusion protein validation, biotin delivery strategies, protein extraction and enrichment, and mass spectrometry-based analysis. We also highlight emerging quantitative and conditional PL approaches that enable dynamic comparison of proxiomes across developmental stages, environmental conditions, and genetic backgrounds. Throughout, we emphasize the importance of rigorous controls, careful terminology, and orthogonal validation to ensure biologically meaningful interpretation of PL datasets. These guidelines aim to standardize experimental design and interpretation, facilitating reproducible and biologically meaningful PL studies in plant systems.