PROJECT SUMMARY/ABSTRACT The challenge addressed by our T-R01 project, the global human cell surface interactome, is to generate maps of 1) the in vitro interactions among the extracellular domains (ECDs) of human cell-surface proteins (CSPs) and “orphan” secreted proteins and 2) the functional effect of these proteins on primary cells from the human immune and nervous systems. There are about 2000 CSP and orphan secreted proteins that are compatible with our in vitro binding and primary cell screening platforms. Creating a map of pairwise interactions of 2200 proteins requires testing 4.8 million interactions. This is beyond the capacity of current in vitro binding methods. To overcome this limitation, we have developed a method to multiplex the in vitro binding screen. The multiplexing technology uses “dye-barcoded” bait beads and high-avidity prey nanoparticles. To assess the functional effects of CSP and secreted proteins on primary human immune and nervous system cells, we will use automated platforms for flow cytometry, imaging, multiplexed single-cell mRNA sequencing, and single-cell spatial transcriptomics. We have developed a method to perform cost effective multiplexed single-cell mRNA sequencing and are developing a cost effective multiplexed single-cell spatial transcriptomics method. To carry out the in vitro binding and primary cell functional screens, we will utilize an in-house built robotic platform that includes liquid handlers and a chain of auxiliary instruments that will carry out the various steps of these methods. The robotic platform includes a robot arm on a rail that moves plates between the liquid handlers and auxiliary devices. The robotic platform is enclosed in an in-house designed BSL2 enclosure. We have developed fully automated methods for all steps of the in vitro binding screen including: 1) subcloning >2200 synthesized genes into a mammalian expression plasmid, 2) plasmid production, quantitation and concentration normalization, 3) transfection of human Expi293 suspension cell cultures, 4) protein purification and quantitation, 5) automated Western blot analysis (ProteinSimple Jess instrument), and 5) all steps of the multiplexed in vitro binding assay. We are currently developing fully automated methods for the primary cell functional screen. Expi293 cells used for protein production grow in deep-well 96-wel plates (96-DWPs) and require specific shaking conditions (3 mm orbital shaking diameter at 1,200 rpm) in a cell culture incubator. These cells provide the workhorse necessary to produce the library of >2200 proteins. As protein stability is often compromised by prolonged storage at 4 C and/or the freeze-thaw process, a high capacity shaking incubator is necessary for production of thousands of proteins over a reasonable timeframe (~2 months), thereby allowing rapid screening following production. Rapid production will ensure maximum protein integrity/function is retained at the time of screening, thereby ...