Biotinylation by antibody recognition—a method for proximity labeling

Abstract

Identification of protein-protein interactions is a major goal of biological research. Despite technical advances over the last two decades, important but still largely unsolved challenges include the high-throughput detection of interactions directly from primary tissue and the identification of interactors of insoluble proteins that form higher-order structures. We have developed a novel, proximity-based labeling approach that uses antibodies to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues. We showed our method to be specific and sensitive by labeling a mitochondrial matrix protein. Next, we used this method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. The ability to detect proximal proteins and putative interactors in intact tissues, and to quantify changes caused by different conditions or in the presence of disease mutations, can provide a new window into cell biology and disease pathogenesis.

The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function

“…Early biochemical studies of the NL employed fractionation of this structure, but this method is limited by the ability to screen for different biological molecules and the amount of starting material required. This problem led to the development/utilization of a number of proximity ligation methods such as Biotin-mediated identification (BioID), DNA adenine methylation-mediated identification (DamID), Tyramide Signal Amplification (TSA) and Biotinylation by antibody recognition (BAR) (Bar et al, 2018;Chen et al, 2018b;Guelen et al, 2008;Roux et al, 2012). The principle behind each of these methods is the enzymatic tagging of proteins and/or nucleic acids in the proximity of the NL with a molecule that is either readily identified or amenable to purification.…”

“…The NL is linked to a number of dynamic cellular activities including chromatin organization, transcription and RNA/protein trafficking through nuclear pores.Our understanding of the NL has been hindered in part by the general insolubility and low extractability of proteins from this region. This has spurred the development of proximity ligation methods that label proteins and/or DNA near the NL for systematic identification (Bar et al, 2018;Chen et al, 2018b;Guelen et al, 2008;Roux et al, 2012). To simplify labeling and improve temporal resolution, we fused APEX2 (Hung et al, 2014;Lam et al, 2015) to the nuclear lamina protein lamin-B1 to map proteins, RNA and DNA associated with the NL.…”

Exploring the protein–protein interaction landscape in plants

“…For instance, proximity labelling with ascorbate peroxidase that can biotinylate neighbouring proteins within minutes provides the additional advantage of temporal resolution (Martell et al, ; Rhee et al, ). In another proximity‐based labelling approach, limitations related to protein fusion can be overcome by using biotinylation by antibody recognition that deposits biotin via a specific antibody onto proteins in close proximity of the target antigen in fixed cells and primary tissues (Bar et al, ). Moreover, in the “lysis‐free” MS‐based method Virotrap, the POI is fused to HIV‐1 Gag, a protein able to trap interacting partners into virus‐like particles, which are subsequently purified from the medium (Eyckerman et al, ; Titeca et al, ).…”

A Novel Mechanism for NF-κB-Activation via IκB-Aggregation: Implications for Hepatic Mallory-denk-body Induced Inflammation

“…By contrast, biotinylation proximity labeling circumvents this limitation by introducing an enzyme to the target protein that can generate distance-constrained reactive biotin molecules to covalently link neighboring proteins, providing a permanent tag that survives purification under harsh conditions for downstream identification [16]. We chose BAR/APEX over BioID (the two popular methods for in vivo biotinylation proximity) because the BAR approach [17] is much more robust as it enables quicker capture of interacting proteins (as the peroxidase mediated biotinylation takes only a few minutes) without the more protracted BioID methodology (requiring 18-24 h tagging time), which could overlook short-term/transient interactants. BAR analyses were performed according to [17].…”

“…We chose BAR/APEX over BioID (the two popular methods for in vivo biotinylation proximity) because the BAR approach [17] is much more robust as it enables quicker capture of interacting proteins (as the peroxidase mediated biotinylation takes only a few minutes) without the more protracted BioID methodology (requiring 18-24 h tagging time), which could overlook short-term/transient interactants. BAR analyses were performed according to [17]. Briefly, two wells of HEK293T cells grown on collagen-coated 6-well plates were first treated as indicated and then fixed with 4% formaldehyde for 10 min at room temperature and permeabilized for 7 min with 0.5% Triton-X in PBS.…”