Capturing Cardiomyocyte Cell-to-Cell Heterogeneity via Shotgun Single Cell Top-Down Proteomics.

Area of Science:

• Proteomics
• Cellular Biology
• Biochemistry

Background:

• Proteins and their diverse proteoforms dictate cellular functions.
• Understanding proteoform heterogeneity at the single-cell level is crucial but analytically challenging.
• Existing methods struggle to resolve complex proteoform variations within individual cells.

Purpose of the Study:

• To develop and validate a single-cell top-down proteomics (SC-TDP) strategy.
• To enable direct and unbiased proteoform profiling from individual cardiomyocytes.
• To investigate proteoform diversity and heterogeneity at the single-cell level in cardiac tissue.

Main Methods:

• Developed a shotgun single-cell top-down proteomics (SC-TDP) approach.
• Applied SC-TDP to profile proteoforms from individual mouse cardiomyocytes.
• Utilized mass spectrometry for direct proteoform analysis.

Main Results:

• Identified 165 distinct proteoforms across 57 proteins from 13 single cardiomyocytes.
• Detected various modifications including phosphorylation, succinylation, and trimethylation.
• Revealed substantial variations in proteoform composition among individual cardiomyocytes, indicating significant cellular heterogeneity.

Conclusions:

• SC-TDP is a powerful tool for uncovering proteoform diversity at the single-cell level.
• The study establishes a new method for analyzing molecular heterogeneity in cardiac tissue.
• This approach provides unprecedented resolution for examining the proteoform landscape underlying cellular identity and physiology.