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

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.