Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

Marion Müller ,Fabienne Gräbnitz,Sebastian N Steiner,Niculò Barandun,John A Robinson,Fabian Wendt,Yang Shen,Martin J Loessner,Milon Mondal,Marc Van Oostrum,James R Prudent,Alexey I Nesvizhskii,Annette Oxenius,Roman C Sarott,Berend Snijder,Jeffrey W Bode,Raphael Hofmann,Yannik Severin,Erick M Carreira,Stefan U Vetterli,Bernd Wollscheid

doi:10.1038/s41467-021-27280-x

Abstract

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.

Highlights

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease
We tailored a liquid-handling robot-assisted processing workflow to automatically capture and proteolytically digest biotinylated proteins within streptavidin-functionalized tips for LC-MS/MS-based analysis eventually enabling the discoverydriven identification and label-free quantification of labeled proteins with excellent reproducibility and at high sample throughput (96-well format)[14]. Based on this optoproteomic workflow, we designed the LUX-MS technology (Fig. 1) where singlet oxygen generator (SOG) are chemically coupled to any ligand of interest and guided to selected surfaceome signaling domains on living cells to enable their light-induced labeling under physiological conditions
The nanoscale organization and extracellular interaction network of the cellular surfaceome is a key mediator in antibody signaling, drug action, hormone function, viral infection, and intercellular communication

Summary

Introduction

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Most recently, localized proximity labeling by antibody-tethered enzymes (APEX26, horseradish peroxidase (HRP), PUP-iT27) or photocatalysts (μMap)[28] were successfully combined with mass spectrometry for the characterization of selected surfaceome landscapes including lipid rafts[29], BCR neighborhoods[30] and intact neuronal synapses[31] in living cells and in fixed tissues[32] Such antibody-based methods are useful in deciphering surface microenvironments of targeted receptors, highly versatile technologies are required to uncover dynamic surfaceome domains that underlie the signaling function of small molecules, biomolecules, viral particles, and complex intercellular receptor interaction networks as formed within immunosynapses of interacting immune cells during T-cell activation.

Methods

Results

Conclusion