Mapping Interactions between Glycans and Glycan-Binding Proteins by Live Cell Proximity Tagging

Studying interactions between glycans and glycan-binding proteins (GBPs) remains challenging due to the transient and weak nature of these interactions. Traditional methods often fail to capture these interactions in live cells without disrupting the native environment. While glycans are commonly presented as glycoprotein conjugates, identifying the specific proteins involved has been limited. Proximity labeling techniques offer a promising solution by enabling covalent tagging of interacting glycoproteins. This approach allows for the enrichment and identification of GBP interactomes in a live-cell setting. However, prior work has not fully addressed the spatial and temporal dynamics of these interactions. This gap motivated the development of a protocol combining proximity labeling with mass spectrometry to study GBP interactions in their natural context. The need for a robust and reproducible method to capture transient glycan-GBP interactions remains unmet in current literature.

This study aimed to develop and validate a method for mapping glycan-GBP interactions in live cells using proximity labeling and mass spectrometry. The transient and noncovalent nature of these interactions makes them difficult to study with conventional techniques. The protocol introduces a fusion protein system involving a GBP and a peroxidase enzyme to enable in situ tagging of glycoprotein interactors. This approach allows for the spatial and temporal capture of GBP interactions without disrupting the cellular environment. The study also sought to establish a reproducible workflow for the purification, characterization, and analysis of these interactions. By integrating fluorescence microscopy and proteomic analysis, the protocol provides a comprehensive framework for studying GBP-glycan interactions. The goal was to overcome limitations in current methods by preserving the native cellular context while enabling robust identification of interacting proteins.

The method involves the construction of fusion proteins combining a GBP of interest with a peroxidase enzyme to enable proximity labeling. The recombinant fusion protein is expressed in cells and characterized for functionality. In situ labeling is performed using a radical-mediated tagging system, which covalently marks interacting glycoproteins. Fluorescence microscopy is used to visualize the labeling process and confirm spatial localization. The labeled proteins are then enriched and analyzed using high-resolution mass spectrometry for proteomic identification. An alternate protocol includes manual purification of the fusion protein using Ni-NTA columns. A second protocol outlines the use of Western blotting to confirm labeling efficiency. A third protocol details the workflow for quantitative MS-based proteomics using tandem mass tags. These protocols collectively allow for the systematic study of GBP-glycan interactions in live cells.

The method successfully enabled the in situ labeling of glycoprotein interactors using a GBP-peroxidase fusion system. Fluorescence microscopy confirmed the spatial localization of the labeling process. Mass spectrometry analysis identified a range of glycoproteins interacting with the GBP galectin-3. The use of tandem mass tags allowed for quantitative proteomic analysis of the captured proteins. The protocol demonstrated robust enrichment of labeled proteins without the need for static isolation. The method preserved the native cellular environment during labeling and analysis. The workflow was validated using multiple protocols, including Ni-NTA purification and Western blotting. The results showed that the method is effective in capturing transient glycan-GBP interactions in live cells.

The study demonstrated that proximity labeling combined with mass spectrometry is a viable method for studying glycan-GBP interactions in live cells. The GBP-peroxidase fusion system enabled spatial and temporal tagging of interacting glycoproteins. The method overcomes limitations of traditional approaches by preserving the native cellular environment. The use of fluorescence microscopy and proteomic analysis confirmed the effectiveness of the protocol. The workflow provides a reproducible framework for identifying GBP interactomes. The authors propose that this approach allows for robust capture of transient interactions without relying on static systems. The method is suitable for studying a range of GBP-glycan interactions in their natural context. The findings suggest that proximity labeling is a valuable tool for glycobiology research.