# Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis

> **NIH NIH R44** · CYTORUM, INC. · 2024 · $948,534

## Abstract

Summary
 T cells are among the most diverse immunological cell types and are central to the pathology of
a broad range of human diseases. This diversity has largely been defined by their differential
expression of cell surface proteins and to a lesser extent, intracellular protein markers, using flow
cytometry. Although significant progress has been made, the field is unnecessarily limited by the lack
of available antibody reagents. Only 900 of the 20,000 known human proteins have validated flow
cytometry reagents, severely limiting the cellular phenotypes and responses that can be measured.
Indeed, recent studies using proteomics (mass spectrometry) and genomics (scRNAseq) have
identified hundreds of proteins that appear to be differentially expressed within T cell subsets,
suggesting that our current definition of T cells using flow cytometry is quite limited. However, other
antibody-based methods such as Western blot have antibodies available for more than half the
proteome. In our Phase I studies we developed the Exocells method that allows flow cytometry
samples to be processed using SDS and heat, similar to Western blot samples. This format makes the
flow cytometry samples amenable to staining with antibodies that are validated to work using Western
blots. This fundamental advance in flow cytometry sample processing promises to dramatically
increase the number of antibodies that can be used in flow cytometry to characterize T cell populations
and cell states. In the Phase II studies, we will further optimize the method and perform assay
qualification in primary cell samples. Using the optimized method, we will expand the existing repertoire
of T cell markers by more than 30%. Finally, using mass cytometry, which is capable of analyzing 40 or
more parameters per cell, we will create a T cell staining panel containing 25 known surface markers
and 15 of the newly discovered intracellular proteins. This panel will be a powerful tool for identification
of novel T cell subsets, for biomarker discovery in various T cell-mediated disease states, and for
analysis of immuno-oncology therapeutics. By significantly increasing the number of flow cytometry
reagents capable of differentially staining T cells, we seek to drive the discovery of the next generation
of T cell subsets and their role in human disease progression and therapeutic efficacy.

## Key facts

- **NIH application ID:** 10890177
- **Project number:** 5R44AI167519-03
- **Recipient organization:** CYTORUM, INC.
- **Principal Investigator:** Peter Krutzik
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $948,534
- **Award type:** 5
- **Project period:** 2022-05-04 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10890177

## Citation

> US National Institutes of Health, RePORTER application 10890177, Proteome Capture in Hydrogel Beads for High Resolution Single Cell Analysis (5R44AI167519-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10890177. Licensed CC0.

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