A chemical proximity labeling platform for mapping the microenvironment and interactome of sialylated glycoproteins

Background

Glycosylation, the enzymatic process that attaches glycans to proteins, is one of the most prevalent post-translational modifications in mammalian cells, affecting over half of all proteins. This modification plays a critical role in determining protein folding, stability, localization, and function, and is intimately involved in a variety of physiological and pathological processes, including immune response, inflammation, and cancer progression. Of particular interest is sialylation, the addition of sialic acid residues to the terminal positions of glycan chains on glycoproteins. Sialic acids are known to mediate key biological interactions, such as cell-cell recognition and signaling, and are implicated in processes like tumor metastasis and immune evasion. Despite their importance, the spatial and functional complexity of sialylated glycoproteins on cell surfaces remains poorly understood, largely due to the lack of high-resolution tools capable of mapping their local molecular environments.

Current approaches to studying glycoprotein microenvironments and interactomes suffer from several significant limitations. Traditional biochemical and proteomic techniques, such as immunoprecipitation and mass spectrometry, often lack the spatial resolution needed to distinguish direct neighbors from more distant interactors, leading to ambiguous or misleading results. Methods like proximity labeling using enzymes (e.g., BioID or APEX) provide some spatial information but are limited by enzyme size, labeling radius, and the need for genetic manipulation, which can perturb native protein function or localization. Additionally, these approaches generally do not offer specificity for particular glycan structures, such as sialylation, making it difficult to dissect the unique roles of sialylated glycoproteins in complex biological contexts. As a result, there is a pressing need for technologies that can selectively and precisely map the immediate molecular environment of sialylated glycoproteins to better understand their roles in health and disease.

Technology

This technology enables high-resolution, proximity-based labeling of sialylated glycoproteins on cell surfaces by leveraging a transition metal catalyst—most commonly iridium—covalently attached to glycoproteins via a derivatized sialic acid linker and click chemistry. The system is metabolically incorporated into living cells, and upon light activation, the catalyst transfers energy to a protein labeling agent (such as a diazirine or azide), generating a highly reactive intermediate. This intermediate covalently tags proteins and biomolecules within a nanometer-scale radius, allowing for precise mapping of the local proteome and interactome surrounding specific sialylated glycoproteins. The labeling agent can be functionalized with markers like biotin, enabling downstream detection through proteomics or imaging. The platform is validated across various cell types and supports workflows such as tandem mass tag (TMT)-based chemoproteomics for quantitative identification of both directly sialylated proteins and their proximal interactors.

What differentiates this technology is its unprecedented spatial resolution and specificity in profiling the microenvironment of sialylated glycoproteins, a class of molecules central to processes like cancer progression, immune evasion, and cell signaling. Unlike traditional proximity labeling methods, this approach allows for tunable control over the labeling radius and intermediate lifetime through chemical design, resulting in higher specificity and efficiency—demonstrated by superior tagging rates compared to conventional probes. The integration of click chemistry ensures modularity and adaptability, while the use of transition metal photocatalysts enables robust, light-triggered activation. The platform’s ability to quantitatively map interactomes and link sialylation to functional changes in transporter activity and cellular metabolism provides powerful new insights into disease mechanisms, making it a versatile tool for both basic research and diagnostic applications.

Benefits

• Enables high-resolution, proximity-based labeling of sialylated glycoproteins on cell surfaces for precise mapping of local proteomes and interactomes.
• Utilizes a transition metal photocatalyst (e.g., iridium) covalently linked via a derivatized sialic acid and click chemistry, ensuring specificity and modularity.
• Generates reactive intermediates with tunable diffusion radius and lifetime, allowing controlled spatial labeling within nanometer-scale proximity.
• Supports downstream detection and quantitative proteomic analysis through incorporation of molecular markers like biotin and tandem mass tags.
• Demonstrates superior labeling efficiency and specificity compared to traditional proximity labeling methods across multiple cell types.
• Reveals biologically relevant interactions, including modulation of membrane transporter functions and ion homeostasis linked to sialylation, especially in cancer cells.
• Facilitates integration of chemical, biological, and analytical workflows for reproducible and adaptable applications in research and diagnostics.

Applications

• Cancer cell surface proteome mapping
• Drug target identification in oncology
• Biomarker discovery for disease diagnostics
• Membrane protein interactome profiling
• Ion transporter function analysis

Additional info

This system utilizes an iridium photocatalyst covalently tethered to cell-surface glycoproteins via sialic acid linkers and click chemistry. Light activation triggers energy transfer to labeling agents, producing reactive intermediates that covalently tag biomolecules within a nanometer-scale radius. This proximity-based approach enables high-resolution mapping of the local proteome and interactome, revealing spatial relationships between glycoproteins and membrane proteins involved in cellular metabolism and ion homeostasis.