# A Global Map of Interactions Among Human Cell Surface Proteins and Secreted Ligands

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2024 · $178,914

## Abstract

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 ...

## Key facts

- **NIH application ID:** 11101052
- **Project number:** 3R01GM150125-03S1
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Kenan Christopher GARCIA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $178,914
- **Award type:** 3
- **Project period:** 2022-09-30 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11101052

## Citation

> US National Institutes of Health, RePORTER application 11101052, A Global Map of Interactions Among Human Cell Surface Proteins and Secreted Ligands (3R01GM150125-03S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/11101052. Licensed CC0.

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