The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate complexes during lysis and enrichment procedures. Advantages of this new approach are illustrated through application to PTP1B interaction networks in models of HER2-positive and Herceptin-resistant breast cancer. We have demonstrated that inhibitors of PTP1B significantly reduced proliferation and viability in cell-based models of acquired and de novo Herceptin resistance in HER2-positive breast cancer. Using differential analysis, comparing substrate-trapping to wild-type PTP1B, we have identified multiple unreported protein targets of PTP1B with established links to HER2-induced signaling and provided internal validation of method specificity through overlap with previously identified substrate candidates. Overall, this versatile approach can be readily integrated with evolving proximity-labeling platforms (TurboID, BioID2, etc.), and is broadly applicable across all PTP family members for the identification of conditional substrate specificities and signaling nodes in models of human disease.

Protein-protein interactions are central to most cellular processes and their dysregulation has been related to the development of various diseases. Proximity-based labeling methods are used to identify the endogenous interaction partners of specific proteins of interest (POIs). The POI is fused to promiscuous enzymes, which generate reactive species in vivo and label proteins in close vicinity. APEX-based proximity labeling techniques utilize an engineered ascorbate peroxidase, which in the presence of H2O2 oxidizes biotin-phenol to short lived biotin-phenoxyl radicals that biotinylate nearby proteins. The biotinylated proteins are enriched by biotin affinity capture and identified by mass spectrometry. We devised an advanced method, RAPIDS, in which the peroxidase is physically separated from the POI and only a rapamycin-induced dimerization using the FRB-FKBP12 system brings the two proteins together. RAPIDS improves the specificity of APEX-based interactome analysis by strictly eliminating false positives. In this chapter, we describe this method in detail, with VAPB as a protein of interest and versions of APEX2 with different subcellular localizations. VAPB localizing to different cellular compartments, the endoplasmic reticulum and the inner nuclear membrane, yielded distinct sets of proximity partners as identified by RAPIDS.

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCE Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

Protein-protein interactions are of paramount importance in regulating normal cardiac physiology. Methodologies to elucidate these interactions in vivo have been limited. Recently, proximity-dependent biotinylation, with the use of BioID, TurboID, and ascorbate peroxidase, has been developed to uncover cellular neighborhoods and novel protein-protein interactions. These cutting-edge techniques have enabled the identification of subcellular localizations of specific proteins and the neighbors or interacting proteins within these subcellular regions. In contrast to classic methods such as affinity purification and subcellular fractionation, these techniques add covalently bound tags in living cells, such that spatial relationships and interaction networks are not disrupted. Recently, these methodologies have been used to identify novel protein-protein interactions relevant to the cardiovascular system. In this review, we discuss the development and current use of proximity biotin-labeling for cardiovascular research.

Protein-protein interactions are critically important for cellular functions, including regulation of ion channels. Ion channels are typically part of large macromolecular complexes that impact their function. These complexes have traditionally been elucidated via standard biochemical techniques including immunoprecipitation, pull-down assays and mass spectrometry. Recently, several methods have been developed to provide a more complete depiction of the microenvironment or \"neighborhood\" of proteins of interest. These new methods, which fall broadly under the category of proximity-dependent labeling techniques, aim to overcome the limitations imposed by antibody-based techniques and mass spectrometry. In this chapter, we describe the use of proximity labeling to elucidate the cardiac CaV1.2 macromolecular complex under basal conditions and after β-adrenergic stimulation. Using these methodologies, we have identified the mechanism underlying adrenergic stimulation of the Ca2+ current in the heart.

One of the most fascinating features of the brain is its ability to adapt to its surroundings. Synaptic plasticity, the dynamic mechanism of functional and structural alterations in synaptic strength, is essential for brain functioning and underlies a variety of processes such as learning and memory. Although the molecular mechanisms underlying such rapid plasticity are not fully understood, a consensus exists on the important role of proteins. The study of these neuronal proteins using neuroproteomics has increased rapidly in the last decades, and advancements in MS-based proteomics have broadened our understanding of neuroplasticity exponentially. In this review, we discuss the trends in MS-based neuroproteomics for the study of synaptic protein-protein interactions and protein signaling dynamics, with a focus on sample types, different labeling and enrichment approaches, and data analysis and interpretation. We highlight studies from the last 5 years, with a focus on synapse structure, composition, functioning, or signaling and finally discuss some recent developments that could further advance the field of neuroproteomics.

The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium and the centriolar satellites, which together regulate cell polarity, signaling, proliferation and motility in cells and thereby development and homeostasis in organisms. Accordingly, deregulation of its structure and functions is implicated in various human diseases including cancer, developmental disorders and neurodegenerative diseases. To better understand these disease connections, the molecular underpinnings of the assembly, maintenance and dynamic adaptations of the centrosome/cilium complex need to be uncovered with exquisite detail. Application of proximity-based labeling methods to the centrosome/cilium complex generated spatial and temporal interaction maps for its components and provided key insights into these questions. In this review, we first describe the structure and cell cycle-linked regulation of the centrosome/cilium complex. Next, we explain the inherent biochemical and temporal limitations in probing the structure and function of the centrosome/cilium complex and describe how proximity-based labeling approaches have addressed them. Finally, we explore current insights into the knowledge we gained from the proximity mapping studies as it pertains to centrosome and cilium biogenesis and systematic characterization of the centrosome, cilium and centriolar satellite interactomes.

BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.