Primary samples, including freshly isolated cells, tissues, and clinical specimens, preserve native physiological states and microenvironments, making them especially valuable for understanding biological systems in health and disease. Proximity labeling (PL) has emerged as a powerful strategy for interrogating molecular interaction networks in situ, but conventional enzyme-based approaches are often difficult to apply to primary samples due to their reliance on genetic manipulation and sustained exogenous expression. Photocatalytic proximity labeling (PPL) provides a non-genetic alternative in which small-molecule photocatalysts activate labeling probes under light irradiation, enabling temporally gated and spatially localized covalent tagging of proximal biomolecules in a wide range of contexts. This review summarizes recent developments in the application of PPL to primary samples, spanning organelle-resolved proteomics, cell surface protein interaction profiling, cell-cell interaction analysis in tissues and in vivo, and emerging immune-engineering applications. While a growing diversity of photocatalytic systems and probe chemistries has been reported, their evaluation and deployment in primary cells and tissues remain at an early stage, reflecting the heightened demands for efficiency, specificity, and robustness in native biological contexts. We discuss how advances in reaction chemistry, catalyst targeting, and long-wavelength activation are beginning to address these challenges, and we outline key opportunities and limitations for extending PPL toward broader use in primary samples. Continued development of primary-oriented photocatalytic toolkits is expected to facilitate more direct interrogation of native biological systems, providing valuable insights into cellular organization, tissue-level communication, and disease-associated molecular remodeling.
Chemical proteomics has emerged as a powerful approach to decipher protein function, interactions, and targeted degradation pathways in complex biological systems. Recent advances in chemical labeling strategies, including activity-based protein profiling (ABPP), proximity labeling (PL), and proteolysis-targeting chimeras (PROTACs), have facilitated a deeper understanding of protein function and interaction networks. First, ABPP employs covalent probes to selectively label active enzymes, uncovering functional proteomics and drug-target interactions. Innovations such as PhosID-ABPP and streamlined cysteine ABPP have improved site-specific quantification and throughput, enabling proteome-wide analysis of enzyme activity and small-molecule interactions. Second, PL enables the characterization of transient protein-protein interactions using enzymatic or chemically triggered approaches. Advances including TurboID and TransitID enhanced the spatiotemporal resolution of PL. Third, PROTACs expand the scope of targeted protein degradation by leveraging the ubiquitin-proteasome system. Collectively, we highlight recent advancements in integrating mass spectrometry (MS) with these methodologies in the field of chemical proteomics.
Protein-protein interactions (PPIs) and spatially restricted molecular contacts govern cellular function, yet many are poorly captured by classical biochemical approaches that rely on cell lysis or stable complex isolation. Proximity labeling (PL) technologies have transformed interactome analysis by enabling covalent tagging of biomolecular neighborhoods directly within intact cells, tissues, and living organisms. By generating short-lived reactive species, PL provides spatially and temporally resolved snapshots of molecular organization under native conditions. Recent advances across enzymatic, chemical, and photocatalytic PL platforms have expanded control over labeling radius, kinetics, and activation, while reducing background and enabling microenvironment-specific targeting. Hybrid genetic-chemical and optogenetic strategies further extend PL beyond mapping toward proximity-based signal amplification and functional interrogation. This review focuses on the most significant methodological and conceptual advances in proximity labeling reported over the past two years, highlighting how these developments have enabled discovery of previously inaccessible interaction networks, including membrane assemblies, chromatin complexes, and in vivo protein microenvironments. We conclude by outlining key challenges and future opportunities for proximity labeling in interactome mapping and amplification.
Cell-cell interactions are fundamental to multicellular organisms, governing development, maintaining tissue homeostasis, and enabling responses to perturbations. Accordingly, the development of effective tools to investigate cell-cell communication in its native context is essential for addressing fundamental biological questions. In recent years, substantial progress has been made toward studying these processes directly in vivo, where cellular dynamics, tissue architecture, and environmental cues are preserved. This review discusses these in vivo advances, focusing on how chemical and synthetic biology tools enable the interrogation and control of cell- and protein-mediated communication in living organisms. We discuss the evolution of these technologies and illustrate how they are being applied to uncover general principles of cellular interaction within complex biological systems.
Studying protein and nucleic acid interactions within chromatin remains challenging, as these events occur in a densely packed and dynamic nuclear environment. Proximity labeling technologies now offer powerful solutions by enabling spatially and temporally resolved characterization of chromatin microenvironments in living cells. Recent advances in labeling chemistry, photocatalytic platforms, and targeting strategies have improved precision and efficiency, broadening their utility across chromatin biology. Here, we review protein-centered proximity labeling approaches-including nanoscale and mesoscale photocatalytic systems, genetically encoded modalities, and antibody-directed labeling-that reveal histone-associated interaction networks and PTM-dependent microenvironments. We also highlight nucleic-acid-centered platforms that use CRISPR guidance, hybridization-based targeting, or structure-specific sensors to map protein complexes at defined genomic loci, diverse RNA species, and noncanonical structures such as G-quadruplexes and R-loops. Together, these technologies are reshaping how chromatin interactomes are measured. We conclude by outlining key challenges and future opportunities that will guide next-generation proximity labeling tools for chromatin research.
Modern proteomic efforts aim to define protein composition and protein-protein interactions within native spatial tissue contexts to understand disease mechanisms and therapeutic responses. Two complementary strategies dominate this effort: physically resolved mapping using high-plex imaging, mass cytometry, or laser-capture microdissection, and chemically defined spatial proteomics based on enzyme or photo-proximity-mediated labeling. Photo-proximity labeling enables nanometer-scale mapping of protein neighborhoods with precise spatial control, overcoming limitations of enzyme-based approaches. This review highlights emerging light-driven spatial proteomic technologies, including targeted photocatalytic labeling and optically guided microproteomics, that combine optical precision with deep proteome coverage to generate high-resolution interactome maps. These methods enable analysis of molecular organization across in vitro and in vivo systems, including formalin-fixed, paraffin-embedded tissues, and reveal disease-relevant molecular reorganization. We discuss recent advances in photo-proteomic strategies that provide mechanistic insight into protein organization in patient tissues and support biomarker discovery in cancer, neurodegeneration, and other complex diseases.
By generating short-lived reactive intermediates that covalently tag nearby biomolecules, proximity labeling (PL) has become a central strategy for spatial proteomics and for probing protein-protein interactions in living systems. However, key aspects of PL performance, including labeling radius, temporal resolution, and biological compatibility, are ultimately governed by how these intermediates are produced, confined, and quenched in situ. Here we use reactive-intermediate generation as an organizing chemical framework to compare major PL modalities. We focus primarily on proteome-centered PL systems, while noting that the same framework can extend to other biomolecular readouts. We discuss peroxide- and oxygen-driven platforms that form phenoxyl radicals and quinone electrophiles, ATP-coupled ligase approaches that transfer activated intermediates to proximal nucleophiles, and light-triggered photocatalytic systems that access carbenes or nitrenes, singlet oxygen or radical reactive oxygen species, and photoredox-uncaged electrophiles. Across these manifolds, we highlight the trade-offs that set operational boundaries and outline design principles for next-generation PL that is quantitative, minimally perturbative, and increasingly in vivo compatible.
Despite major advances in biomedical research, dissecting disease-relevant molecular pathways remains challenging due to pathway redundancy, transient protein interactions, and the limited spatiotemporal precision of existing tools. Genetic code expansion (GCE) addresses these limitations by enabling site-specific incorporation of noncanonical amino acids that endow proteins with novel chemical, photophysical, or regulatory properties directly in living systems. This capability provides unique access to dynamic protein interactions, post-translational modifications, and signaling events in cellular environments. Here, we highlight recent advances in GCE that are particularly adapted to studying disease biology in increasingly physiologically relevant contexts, discuss key challenges limiting broader implementation, and outline emerging methodologies that position this technology as a transformative synthetic biology platform for mechanistic dissection of disease processes.
Covalent lysine targeting is an active frontier in drug discovery and chemical biology as the field extends beyond covalent binding to cysteines. Targeting abundant and functionally diverse lysine residues presents unique opportunities for developing chemical strategies of protein modulation, with the potential to address previously unexplored areas of the proteome. In this review, we highlight recent key advancements in the field, emphasizing electrophilic chemistries, as well as various discovery methods for novel ligands. We review strategies for mapping lysine 'ligandability' and covalent lysine-targeting probe discovery. While the majority of reported covalent lysine binders are still designed in a 'ligand-first' approach, examples of 'electrophile-first' probes are now being reported. From a kinetic perspective, advanced lysine-covalent inhibitors are now approaching second-order reaction rates of Food and Drug Administration (FDA)-approved cysteine-targeting covalent drugs, underscoring the potential of lysine-targeting as a strategy for drug development.
In vivo chemical cross-linking mass spectrometry (XL-MS) has emerged as a powerful technique for high-throughput, proteome-wide mapping of intramolecular conformations and intermolecular interactions of protein complexes in living cells. By providing distance constraints between specific residues, XL-MS enables the characterization of protein conformations and interaction networks under near-physiological conditions, greatly facilitating the analysis of biomacromolecular functions and regulatory mechanisms. The information obtained from cross-linking is particularly valuable at the systems level, and its value continues to increase with improvements in the density of cross-link identification, the precision of distance constraints, and the spatiotemporal resolution. In recent years, advances in cross-linker design, cross-linked peptide enrichment methods, mass spectrometry analysis, and artificial intelligence-assisted data analysis have significantly expanded the capabilities of in vivo XL-MS. This article systematically reviews the latest progress in in vivo XL-MS for protein conformation and interaction network analysis, highlights its unique advantages, discusses current technical challenges, and explores further development.
Site-specific ubiquitylation of nucleosomal histones, catalyzed by E3 ubiquitin ligase, plays a pivotal role in chromatin-templated processes, including transcriptional activation, gene silencing, and DNA damage repair. However, the inherently transient and dynamic interactions between the ubiquitin enzymes and the nucleosome substrate during the ubiquitylation cascade pose significant challenges to stabilizing functional complexes for structural and biochemical interrogation, thereby impeding mechanistic dissection. Recent advances in chemical biology strategies have emerged as powerful tools for resolving ternary ubiquitylation complexes of E3 ligase, E2∼Ub, and substrate. In this review, we systematically survey these innovative chemical approaches for trapping labile nucleosome ubiquitylation intermediates and consolidate the mechanistic insights into chromatin ubiquitylation biology.
Super-resolution proximity labeling (SR-PL) advances spatial proteomics beyond conventional protein-level enrichment, enabling residue-resolved analysis of subcellular organization in living cells. Conventional proximity labeling relies on streptavidin-based capture and on-bead digestion, producing protein-centric readouts with limited structural insight. In contrast, SR-PL directly recovers biotinylated peptides and identifies labeled amino acid residues by LC-MS/MS. These site-specific labels serve as direct evidence of proximity, allowing for the precise mapping of protein surfaces, solvent accessibility and interaction interfaces. By linking spatial proximity to specific structural features, SR-PL enables mechanistic interpretation of spatial proteomic data and reframes proximity labeling as a structure-informed analytical framework. Recent advances in affinity capture strategies-including engineered probes, reversible affinity matrices, and optimized antibody reagents-have improved selective enrichment and gentle peptide release while reducing background contamination. Together, these developments position SR-PL for broad applications such as membrane topology mapping, organelle contact site analysis, and ligand-dependent interactions.
Epilepsy is a widespread neurological disorder characterized by recurrent seizures resulting from abnormal electrical activity in the brain. The origin and severity of these seizures vary depending on the brain regions involved. A key factor influencing seizure susceptibility is vitamin B6, an essential cofactor in neurotransmitter metabolism, where both deficiency and excess can lead to adverse health outcomes. Vitamin B6 homeostasis is regulated in part by Δ1-piperideine-6-carboxylic acid (P6C), a metabolite of lysine catabolism that irreversibly inactivates B6 vitamers. Notably, plants synthesize vitamin B6 de novo and generate P6C during lysine breakdown. Similar to its effects in humans, increase in P6C disrupts cellular metabolism and weakens immune responses in plants. These striking parallels are attributable to conserved metabolic pathways derived from ancient bacterial enzymes that govern vitamin B6 biosynthesis and lysine degradation. This review explores the biochemical basis of epilepsy and highlights the role of plant-derived nutrients, particularly vitamin B6 in supporting human neurological health, while offering evolutionary perspectives on shared metabolic vulnerabilities.
Synthetic biology aims to re-engineer living cells into autonomous computational chassis capable of executing sophisticated biological tasks. Within this framework, programmable nucleic acid-based logic networks have emerged as a versatile molecular control layer for constructing intelligent cellular systems, offering unparalleled precision, orthogonality, and interoperability. Here, we highlight recent advances in molecular programming, focusing on the integration of synthetic DNA circuits within cellular environments to achieve logic-gated control of cellular functions. We first delineate the fundamental building blocks-including strand displacement, logic gates, amplifiers and neuromorphic architectures-and then examine strategies for interfacing these components with endogenous pathways. The field is currently witnessing a paradigm shift from ex vivo demonstration to in situ functional implementation, driven by the maturation of nucleic acid-based engineering within synthetic biology. Ultimately, these programmable molecular controllers enable the rational design of cellular behaviors, paving the way for next-generation precision therapeutics and autonomous biomanufacturing.
The field of protein therapeutics has evolved from native biologics to precision-engineered multi-functional therapeutic agents, overcoming inherent limitations such as structural instability, rapid clearance, and immunogenicity. While early strategies, including PEGylation and albumin fusion, and the emergence of antibody-drug conjugates (ADCs), have demonstrated clinical value, challenges remain in improving the site-specificity of modifications, suppressing structural heterogeneity, adjusting linker stability, and broadening payload diversity for protein therapeutics. Recent advances in bioconjugation techniques, such as chemoselective reactions, enzymatic labeling, glycan engineering, and genetic code expansion, now enable more site-specific and homogeneous modifications, achieving enhancements in batch consistency, controlled payload release, and multi-functionality. This review highlights key developments over the past two years across three categories of modified protein therapeutics: ADCs, cytokines, and proximity-enabled covalent therapeutics. Looking forward, we outline future directions focused on scalable site-specific platforms, immunogenicity management, and delivery optimization, increasingly propelled by artificial intelligence-aided protein design and reaction manipulation.
The integration of proximity labeling (PL) and advanced mass spectrometry-based proteomics is a robust framework for mapping protein-protein interaction (PPI) networks and local protein inventories in the crowded multimolecular environment of live cells. Over the last decade, numerous PL technologies such as biotin identification (BioID), ascorbate peroxidase (APEX) etc. using engineered enzymes or synthetic photocatalysts have been developed and successfully used in cell-based experiments. However, the application of such technologies beyond cultured cells, (i.e. in more complicated tissues or in vivo) remains challenging. In this review, we summarize the current issues in applying PL methods in vivo and highlight recent studies that could provide breakthroughs to overcome the existing limitations and expand the application of PL to tissues and in vivo.
Protein binders are fundamental tools in chemical biology, key components of biotechnologies, and the foundation of biologics-based medicines. However, no binder discovery method has achieved the fidelity, speed, and cost-effectiveness required for routine laboratory use. We argue that the field stands at an inflection point. In vitro display platforms are now mature but have continued evolving through macrocyclization methods, covalent chemistries, and massive quantitative datasets-expanding chemical space while deepening mechanistic understanding of selection outcomes. Computational de novo design-which has exploded in power over the past few years-is transitioning from methods requiring specialized expertise to more accessible platforms. Finally, in vivo directed evolution technologies are emerging as a critical frontier, which may deliver the throughput and fidelity needed to finally democratize binder discovery. This review summarizes these advances and points toward a future where binder generation is fast, accessible, and a key driver of the next phase of biological research.
Proximity labeling (PL) has emerged as a powerful technology for mapping subcellular compositions and molecular interactions. This approach employs promiscuous enzymes that generate reactive species to tag endogenous biomolecules, which are then identified by mass spectrometry or sequencing. However, conventional PL methods-such as peroxidases, biotin ligases, and photocatalytic systems-face significant limitations for in vivo applications, hindering their use in native biological contexts. In this review, we first summarize the application of peroxidases and biotin ligases in living animals, highlighting how they have provided insights into cell surface proteomes and cell type-specific secretion, despite their constraints. We then explore recent advances in in vivo-compatible PL technologies, including novel enzymes like tyrosinase, laccase, and lipoic acid ligase, as well as innovative photocatalytic strategies activated by near-infrared light, ultrasound, or bioluminescence. These emerging tools hold great potential to expand spatial multi-omics from cellular systems to living organisms.
Enzyme cascades enable streamlined multi-step biocatalysis in one-pot systems, offering remarkable efficiency, selectivity, and sustainability compared to traditional chemical synthesis. This review highlights recent advances across three major directions: (i) efficient synthesis of bulk and chiral chemicals, including alcohol-to-amine conversion, polymer precursor biosynthesis, and non-canonical amino acid production; (ii) generation of complex pharmaceutical building blocks, exemplified by the synthesis of nucleotides, alkaloids, isoprenoids and their analogues; and (iii) integration of new-to-nature enzymatic reactions into metabolic pathways exemplified for engineered carbene-transferring P450s, artificial metalloenzymes, and photocatalytic active enzymes within microbial cell factories. These advances, driven by reaction route design, key enzyme engineering, and pathway/cell optimization, position enzyme cascades as transformative and versatile platforms for sustainable biomanufacturing across pharmaceuticals, chemicals, and materials.
Enzymes are typically understood as agents of biological fidelity, but this characterization obscures their growing role as programmable catalysts for molecular invention. In this review, we examine the emerging logic of biocatalyzed nucleic acid synthesis beyond the genetic canon, organizing recent progress into two complementary paradigms: template-dependent and template-independent enzymatic construction. Rather than cataloguing individual modifications, we emphasize the organizing principles that enable enzymes to operate outside their evolutionary remit, including the roles of templating, substrate modularity, steric gating, and reaction cycling in preserving sequence information while expanding chemical scope. We highlight how these principles enable the synthesis of therapeutically relevant oligonucleotides at scale, support the exploration of alternative genetic systems, and allow chemical functionality to be encoded directly into informational polymers.