# 3D Proteomics at Single Cell Resolution with Covalent Protein Painting (CPP)

> **NIH NIH R33** · SCRIPPS RESEARCH INSTITUTE, THE · 2024 · $240,092

## Abstract

Project Summary/Abstract
Somatic mutations alter the folding and therewith function of oncogene and tumor suppressor proteins. The
alterations in protein structure drive malignant transformation of cells. While structural changes of somatic
mutated proteins have been extensively characterized with high spatial resolution, conformational alterations of
other nonmutated effector proteins in the cancer proteome remain elusive. The heterogeneity of protein
conformations and protein-protein interactions in tumors at the single cell level remains unknown, and it is unclear
whether tumor cells with the same genetic background differ in protein conformation or protein-protein
interactions in non-mutated proteins.
Here, we propose to implement a new mass spectrometry-based protein footprinting technique to measure
protein conformations in single cells. We recently developed Covalent Protein Painting which infers protein
structural information with a chemical protein footprinting technique that surveys the chemical reactivity of lysine
residues in proteins to determine alterations in protein conformation or protein-protein interactions in a
proteome. We propose new versions of CPP that will overcome the current limitations in sample preparation
and analysis of single cell proteomes (CPP-SCP). We will quantify lysine site accessibility in single cells with
CPP-SCP, and we will establish bioTMT-CPP-SCP, a variation of the CPP-SCP method that can directly
compare changes in the 3D proteome between several (>10) single cells with high sensitivity. We propose to
analyze intact single cells that are isolated from two cancer cell lines and from murine tumor tissues. Our goal
is to find out whether measurements at single cell level recapitulate structural alterations that we observed in
bulk tumor samples. Specifically, it remains unclear whether aberrant protein conformations per protein are
observed at equal abundance across all cells or if they are confined to a subset of cells in a cancer cell line or
tumor. We hope that CPP-SCP will allow us to differentiate between intra- and intercellular variation in protein
conformation and folding.

## Key facts

- **NIH application ID:** 10917675
- **Project number:** 1R33CA272339-01A1
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** John R Yates III
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $240,092
- **Award type:** 1
- **Project period:** 2024-08-14 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10917675

## Citation

> US National Institutes of Health, RePORTER application 10917675, 3D Proteomics at Single Cell Resolution with Covalent Protein Painting (CPP) (1R33CA272339-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10917675. Licensed CC0.

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