Molecular Pixelation - dissecting the spatial organization of 80 surface proteins in single immune cells

Abstract Immune cell function is dependent on the activity of membrane proteins. Most widely-used cell analysis methods, flow cytometry and scRNAseq, directly or indirectly assess membrane protein abundance, yet provide no information about their spatial distribution. The gold standard technology for spatial protein analysis, fluorescence microscopy, has revealed that the spatial organization of membrane proteins is in fact of importance for their function, as exemplified by the immune synapse and uropods in migrating cells. Limited multiplexing and throughput scalability of microscopy, reduces its potential for forefront spatial proteomics discoveries, highlighting the need for new innovative methods. Here, we present Molecular Pixelation (MPX), an advanced DNA barcode-based method for high-multiplex spatial analysis of proteins in single cells. Combining an NGS-based readout with our dedicated software, Pixelator, MPX enables the reconstruction of 3D cell-surface maps of 80 proteins on over 1000 individual cells, simultaneously. We applied MPX to compare the surface proteome of single resting and in-vitro activated T cells, highlighting differences in protein abundance, as well as revealing dramatic spatial protein re-organization, both in terms of supramolecular clustering and protein-protein co-localization. These results demonstrate that MPX has the potential to pave the way for expanding our understanding of cell surface protein architecture and its role in T cell activation.