Significance: Most of the thousands of proteins that comprise a human cell have specific subcellular localization patterns essential for their function. “Proximity labeling” (PL) is a method for mapping the localization of endogenous cellular proteins on a proteome-wide scale. To improve the specificity and versatility of PL, we developed split-TurboID, a promiscuous biotinylating enzyme split into two inactive fragments. The fragments are coexpressed in cells and brought together by a drug, protein–protein interaction, or organelle contact to reconstitute TurboID enzymatic activity. We used split-TurboID to map the protein composition of endoplasmic reticulum–mitochondria contact sites, which are essential for mitochondrial fission, lipid biosynthesis, and calcium signaling. For conditional or higher-specificity PL, split-TurboID may be a valuable tool for biological discovery.

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle–membrane contact sites in live cells.

Deciphering the sub-compartmental location of a given protein of interest may help explain its physiological function, but it can be challenging to do using optical or biochemical methods. Imaging with electron microscopy (EM) can provide highly resolved mapping of proteins; however, EM requires complex sample preparation and a specialized facility. Here, we use engineered ascorbate peroxidase (APEX)-generated molecular labeling patterns to provide information regarding intracellular microenvironments in living cells. Using APEX labeling of specific proteins, we uncovered subcellular localization at sub-compartmental resolution and successfully elucidated the membrane protein topology of HMOX1 and sub-mitochondrial localization of recently identified mitochondrial proteins. This method can be expanded to confirm sub-mitochondrial localization and membrane topologies of previously identified mitochondrial proteins.

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describeiSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validateiSLET in a whole-body system, we expressiSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liveriSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together,iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Proximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.