Proximity labeling methods enable the identification of proteins in the vicinity of a protein of interest in living cells. Among them, APEX2 proximity is a powerful method to spatiotemporally define in vivo \"proxisomes\" in dynamic bacterial protein systems. Here we describe a standardized APEX2 proximity labeling protocol and possible adaptations to capture protein partners in native conditions.

Cellular compartmentalization of proteins and protein complex formation allow cells to tightly control biological processes. Therefore, understanding the subcellular localization and interactions of a specific protein is crucial to uncover its biological function. The advent of proximity labeling (PL) has reshaped cellular proteomics in infection biology. PL utilizes a genetically modified enzyme that generates a \"labeling cloud\" by covalently labeling proteins in close proximity to the enzyme. Fusion of a PL enzyme to a specific antibody or a \"bait\" protein of interest in combination with affinity enrichment mass spectrometry (AE-MS) enables the isolation and identification of the cellular proximity proteome, or proxisome. This powerful methodology has been paramount for the mapping of membrane or membraneless organelles as well as for the understanding of hard-to-purify protein complexes, such as those of transmembrane proteins. Unsurprisingly, more and more infection biology research groups have recognized the potential of PL for the identification of host-pathogen interactions. In this chapter, we introduce the enzymes commonly used for PL labeling as well as recent promising advancements and summarize the major achievements in organelle mapping and nucleic acid PL. Moreover, we comprehensively describe the research on host-pathogen interactions using PL, giving special attention to studies in the field of virology.

Mammals contain over 200 different cell types, yet nearly all have the same genomic DNA sequence. It is a key question in biology how the genetic instructions in DNA are selectively interpreted by cells to specify various transcriptional programs and therefore cellular identity. The structural and functional organization of chromatin governs the transcriptional state of individual genes. To understand how genomic loci adopt different levels of gene expression, it is critical to characterize all local chromatin factors as well as long-range interactions in the 3D nuclear compartment. Much of our current knowledge regarding protein interactions in a chromatin context is based on affinity purification of chromatin components coupled to mass spectrometry (AP-MS). AP-MS has been invaluable to map strong protein-protein interactions in the nucleus. However, the interaction is detected after cell lysis and biochemical enrichment, allowing for loss or gain of false positive or negative interaction partners. Recently, proximity-dependent labeling methods have emerged as powerful tools for studying chromatin in its native context. These methods take advantage of engineered enzymes that are fused to a chromatin factor of interest and can directly label all factors in proximity. Subsequent pull-down assays followed by mass spectrometry or sequencing approaches provide a comprehensive snapshot of the proximal chromatin interactome. By combining this method with dCas9, this approach can also be extended to study chromatin at specific genomic loci. Here, we review and compare current proximity-labeling approaches available for studying chromatin, with a particular focus on new emerging technologies that can provide important insights into the transcriptional and chromatin interaction networks essential for cellular identity.

The development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein-protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity-tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest (POI) to these enzymes allows the definition of an unbiased \"proxisome,\" that is, all of the proteins in interaction or in close vicinity of the POI. Here, the different proximity-labeling tools available are described and comprehensive comparison to discuss advantages and limitations is provided.