Xu et al. Cell Biosci (2021) 11:27 https://doi.org/10.1186/s13578-021-00542-3 Cell & Bioscience REVIEW Open Access In vivo interactome profling by enzyme‐ catalyzed proximity labeling Yangfan Xu1,2,3, Xianqun Fan2,3* and Yang Hu1* Abstract Enzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the protein-protein interaction networks, dissect complex biological processes, and character- ize the subcellular proteome in a more physiological setting than before. The enzymatic tags are being upgraded to improve temporal and spatial resolution and obtain faster catalytic dynamics and higher catalytic efciency. In vivo application of PL integrated with other state of the art techniques has recently been adapted in live animals and plants, allowing questions to be addressed that were previously inaccessible. It is timely to summarize the current state of PL-dependent interactome studies and their potential applications. We will focus on in vivo uses of newer ver- sions of PL and highlight critical considerations for successful in vivo PL experiments that will provide novel insights into the protein interactome in the context of human diseases. Keywords: Proximity labeling, Protein interactome, APEX, BioID, TurboID Background manipulation and subcellular fractionation for enrich- Proteins generally form complexes, organelles or other ment; these processes are associated with low valida- assemblies and create interacting networks that are tion rates and high false positive rates [4, 5]. Utilization essential for cellular structure and functional integrity. of AP-based methods requires that cells frst being lysed Te execution of biological function and progression to release the bait protein for purifcation, which poses of human disease are intimately tied to protein-protein signifcant challenges to faithfully preserving its in vivo interactions (PPIs), which are characterized by proximity, interacting status. Yeast two-hybridization assay can afnity and duration [1]. Exploration of PPIs underlying only suggest the potential interaction of two proteins, intricate cellular signaling and regulatory mechanisms rather than the interaction that actually takes place has required eforts to circumvent the technical defects in vivo [5]. Fluorescence-based techniques are most suit- in many of the traditional approaches. Te interactome able for validating the candidate interacting partners mapping methods commonly employed are afnity of a given protein, particularly in such applications as a purifcation-mass spectrometry (AP-MS) and yeast two- high-throughput drug screening platform, rather than hybridization [2, 3]. However, these methods very often for identifying novel partners [6, 7]. Te lack of efective fail to reveal in vivo PPIs because they require ex vivo tools to acquire accurate information about protein dis- tribution, protein partners, and protein complex compo- sition remains a major challenge in these felds [8]. *Correspondence: [email protected]; [email protected] In the past decade, enzyme-catalyzed proximity labe- 1 Department of Ophthalmology, Stanford University School of Medicine, ling (PL) has developed as a novel alternative method Palo Alto, CA 94304, USA 2 Department of Ophthalmology, Ninth People’s Hospital, Shanghai to label and capture not only the proteins that interact JiaoTong University School of Medicine, Shanghai, People’s Republic directly with the protein of interest (POI), but also the of China proteins in proximity to the POI [9–11]. In a PL sys- Full list of author information is available at the end of the article tem, a promiscuous labeling enzyme is fused in frame © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xu et al. Cell Biosci (2021) 11:27 Page 2 of 9 to the POI or subcellular compartment marker proteins utilization of PL methods integrated with other sophis- in living cells. Enzymatic catalyzation will covert an ticated approaches to profle protein interactome with inert substrate into a reactive but short-lived intermedi- high confdence. ate, which will then covalently label the nearby biomol- ecules (proteins, RNA and DNA) in a promiscuous and The APEX and HRP system proximity-dependent manner. Because in most cases APEX is a monomeric 28 kDa ascorbate peroxidase that the small-molecule substrate for labeling contains bio- catalyzes the oxidative polymerization and local depo- tin moiety, the biotinylated proteins can be selectively sition of diaminobenzidine (DAB) under harsh treat- enriched by afnity purifcation using neutravidin or ment conditions; DAB can then be stained with the streptavidin coated magnetic or agarose beads. Strepta- electron-dense OsO4 to generate strong contrast for vidin shows a stronger intrinsic binding afnity toward electron microscopy (EM) imaging in mammalian orga- biotin and a lower nonspecifc binding than neutravidin nelles [33]. In living cells, exogenous biotin-phenol (BP) [12, 13]. Due to higher reproducibility, purity-specifcity can be added and catalyzed by APEX in the presence of and ease of use, magnetic beads have become the pre- hydrogen peroxide (H2O2) to produce biotin-phenoxyl ferred support for small-scale experiments, whereas aga- intermediate; biotin-phenoxyl can covalently react with rose beads are more economical when large amounts of electron-rich amino acids, such as tyrosine, in proteins in purifed targeting biomolecules are required. Background the neighborhood [34]. APEX was therefore adopted for contamination is likely to be small because avidin-biotin PL (Fig. 1a, upper panel) [34–36]. One of the major limi- interaction can withstand harsh and stringent purifca- tations of APEX is its relatively low cellular activity and tion conditions and the endogenous biotinylation is rela- sensitivity, which may arise from its sub-optimal fold- tively low in mammalian cells [11]. Te purifed proteins ing/stability, poor heme binding, or some combination are subsequently identifed by high-throughput liquid of these factors [35]. Tus, in order to provide sufcient chromatography coupled to mass spectrometry (LC-MS/ biotinylated proteins for subsequent MS identifcation, MS). Another superior feature of PL is that it can capture higher amount of total protein extracts are typically transient or weak interactions that are often overlooked required. APEX2, which has higher catalytic activity and by conventional AP approaches. Terefore, this tech- sensitivity, was later developed through direct evolution nique facilitates the sensitive, specifc and timely detec- [35] and has been successfully used to determine inter- tion of the interactome of a POI in vivo, which is critical actomes in living cells [37, 38]. Because APEX2 can also for understanding its broad molecular functions quickly, directly biotinylate guanosine in RNAs, APEX-PL has simply and reliably. Accumulating publications have been combined with RNA sequencing (APEX-seq) to broadened the range of the bait proteins, from nuclear determine subcellular transcriptomes [19]. Additionally, membrane proteins and transcriptional factors to ubiqui- APEX2 has been tagged to human telomerase RNA to tin ligases [9, 14, 15], and, recently, even to RNA-protein profle its interactome on a one-minute time scale [18]. interactions [16–20]. Apart from the traditional APEX2 substrate biotin-phe- In addition to its extensive use in cultured mamma- nol, a clickable substrate, alkyne-phenol (Alk-Ph), was lian cells [9, 14, 21, 22], PL has been rapidly adapted for recently shown to improve membrane permeability and in vivo application in a wide variety of research projects enhance labeling efciency in intact yeast cells, which and models, including yeast [23, 24], plant protoplasts enables spatially restricted proteome and transcriptome [25, 26], parasites [27–29], mouse [30, 31], fies and profling in yeast [39]. Tese successful applications worms [32]. In this review, we will focus on the evolution demonstrate higher spatial and temporal resolution of of powerful PL approaches and the important considera- APEX-based PL, which is especially suitable for detec- tions for PL experimental design, especially the in vivo tion of dynamic shifts in interactomes. However, the (See fgure on next page.) Fig. 1 Schematic diagram of enzyme-catalytic proximity labeling approaches. a In a standard enzyme-catalytic proximity labeling system, ascorbate peroxidase (e.g., APEX or APEX2) in the presence of H2O2 catalyzes the one-electron oxidation