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

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.


Field-specific reporting
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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. For experiments including mass spectrometry, sample size was not predetermined by statistical methods for relative quantitative mass spectrometry experiments. However protein quantities were modeled based on the intensity of at least two peptides using Tukey's median polish method ensuring robust label-free quantification that allows for statistical testing with sample size at least n=3 that is commonly employed for quantitative proteomics investigations. For the single cell chemosensitivity screen, 10 technical replicates were analysed per time point based on the previously established pharmacoscopy workflow.
Peptide identifications mapping to decoy or contaminant proteins or internal reference peptides were excluded from further analysis. Additionally, peptides were filtered based on MS/MS identification score to ensure a false discovery rate of < 1%. Outliers based on principal component analysis were removed to retain minimally two biological replicates per condition.
Overall, the anti-CD20 antibody-guided LUX-MS experiments were performed three times with increased replication and demonstrated the reproducibility of the optoproteomic workflow. Labeling and identification of extracellular protein interactions by LUX-MS was validated using bioorthogonal methods such as flow cytometry and confocal microscopy, and literature-based knowledge mining, respectively. Furthermore, small molecule, biomolecule, pathogen and immunogen guided LUX-MS was performed in at least two independent experiments with high overlap of identified proximity candidates. Cell surface localization of phage-LUX identified Listeria monocytogenes proteins was verified in two independent experiments using lysozyme-treated and untreated bacteria. Single cell chemosensitivitiy screen was performed with 10 technical replicates per time point (24 and 48 h of treatment).
This study consists of multiple independent (LUX-MS) experiments with a smaller number of sample (n < 10) that were sequentially acquired by LC-MS/MS thereby minimizing technical variance and obviating the need for randomization.
Investigators were not blinded to allocation of biological samples as the results of the mass spectrometry experiments are of technical nature and not prone to a potential observer bias. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
All commercial antibodies (except Alexa Fluor 555 anti-HA tag monoclonal antibody) were quality tested by flow cytometric analysis by the manufacturer. Alexa Fluor 555 anti-HA tag monoclonal antibody was quality tested by western blot and immunocytochemistry analysis by the manufacturer. The in-house developed anti-CD38 antibody was independently validated by Centrose LLC. Cell surface protein interactions on previously established and commonly used cell lines were investigated using LUX-MS and revealed cell type specific surface markers. No further authentication was applied.

Patient
In the context of this study, cell lines were not specifically tested for Mycoplasma contamination.
No commonly misidentified cell lines were used in this study.
No field collected samples were used in the study.
All animal experiments were performed in accordance with institutional policies and Swiss federal regulations, following guidelines and being approved by the veterinary office of the Canton of Zürich (animal experimental permissions: 115/2017).
FACS experiments in this study used human, murine and bacterial cells. Sample preparation is described in detail in the following methods sections: Antibody-guided surfaceome nanoscale mapping, Synthesis of singlet oxygen generator-coupled cardiac glycoside CG1, Identification of small molecule drug-targeted surfaceome structures, Bacteriophage-guided