Sebastian Steiner

Decoding functional surfaceome protein-protein interactions using light-induced cross-linking sites and HyPhoX

Sebastian N. Steiner1,2 , Clinton Yu3 , André Zwicky4 , Alexander Leitner5 , Jeffrey W. Bode4 , Lan Huang3, Bernd Wollscheid1,2

1 Institute of Translational Medicine (ITM), Department of Health Sciences and Technology (D-HEST), ETH
Zürich, Zürich, Switzerland
2 Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland
3 Department of Physiology & Biophysics, University of California, Irvine, California, USA
4 Laboratory for Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich,
5 Institute of Molecular Systems Biology, Department of Biology, ETH Zurich, 8093 Zürich, Switzerland

Introduction: Not single proteins, but protein communities and protein-protein interactions (PPIs) within the surfaceome of human cells enable complex cellular signaling in response to environmental stimuli. Recent proximity-based protein tagging technology advancements enable the discovery of surfaceome-residing protein communities. Cross-linking of surfaceome-residing complexes can provide orthogonal (functional) information about the surfaceome architecture beyond the general information about proximal protein neighborhoods. Typical in vivo cross-linking approaches utilize chemistries addressing cross-linking sites already available within proteins and generally lack subcellular specificity. Here we developed a new cross-linking strategy, which is utilizing newly light-induced cross-linkable sites on cell surface proteins in combination with a novel tri-functional cross-linker molecule, termed HyPhoX. This Cell Surface Interaction MS (CSI-MS) technology enables the detection of localized surfaceome signaling architecture.

Methods: CSI-MS combines the utilization of light-induced cross-linking sites on cell surface receptors, chemical cross-linking, and mass spectrometry. In the CSI-MS strategy, we generate so-called kodecytes by coating the cell surface of human B lymphocytes with lipids carrying Singlet Oxygen Generators (SOGs). The SOGs enable the light-controlled photo-oxidation of proximal cell surface proteins. Alternatively, SOGs can also be targeted to a specific cell surface receptor using antibodies/ligands, as shown with the LUX-MS strategy, enabling targeted cross-linking of receptor communities. Subsequently, a short illumination of SOGs leads to the oxidation of Histidines within cell surface proteins which can then be used as “induced cross-linkable sites” for selective protein-protein cross-linking of receptor complexes and receptor interaction communities.

Preliminary data: First, we assessed the efficiency of our light-mediated cross-linkable site induction on B lymphocytes. Using imaging and flow cytometry, we observed consistent SOG coating of cells and, consequently global induction of cross-linkable sites on cell surface receptors within 30 seconds of illumination. Upon MS analysis, 205 cell surface proteins were identified bearing light-induced cross-linkable sites. Knowing that we can utilize controlled illumination to induce new chemical moieties at the cell surface that can be used for subsequent chemical cross-linking of cell surface proteins, we set out to develop a new cross-linker molecule targeting these specific sites. This novel tri-functional cross-linker molecule has an IMAC-enrichable PhoX-based affinity handle (Steigenberger et al., 2019) and two reactive hydrazide moieties for targeting the light-introduced Histidine modifications in cell surface proteins. We further incorporated in the design of the cross-linker an MS-cleavable feature that generates signature ions during MS acquisition simplifying identification of spectra containing cross-links and targeted data analysis. Using model peptides and proteins, we confirmed the reactivity of our cross-linker molecule, MS-cleavability, MS cross-linker fragmentation mechanisms, and the detection of signature ions. The fragmentation pattern was used to establish a cross-linker specific search strategy which improves search efficiency for surfaceome wide cross-linking studies. The CSI-MS strategy is currently being tested in pilot experiments to map surfaceome PPIs on B lymphocytes and receptor CRISPR’ed B cells to provide functional insights into surfaceome signaling architecture and residing protein signaling complexes.

Novel aspect: CSI-MS is a strategy enabling the identification of stable cell surface protein communities/signaling complexes/PPis utilizing tri-functional HyPhoX cross-linker and light-inducable cross-linking sites.

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