Highly Sensitive Spatial Proteomics with Multicolor Cleavable Fluorescent Tyramide

Area of Science:

• Biochemistry
• Cell Biology
• Proteomics

Background:

• High-resolution single-cell spatial proteomics provides critical insights into biological systems.
• Current multiplexed protein imaging platforms have limitations in sensitivity, multiplexing capacity, and technical complexity.

Purpose of the Study:

• To develop a highly sensitive spatial proteomics approach for multiplexed protein profiling.
• To overcome the limitations of existing protein imaging platforms.

Main Methods:

• Utilized multicolor cleavable fluorescent tyramide and off-the-shelf antibodies.
• Employed horseradish peroxidase (HRP) for enzymatic fluorophore deposition and reiterative cycles of labeling, imaging, and cleavage.
• Applied the method to human formalin-fixed paraffin-embedded (FFPE) tonsil tissue.

Main Results:

• Quantified 38 proteins in FFPE tonsil tissue, achieving the highest multiplexing capacity with tyramide signal amplification (TSA) methods to date.
• Analyzed approximately 500,000 individual cells, identifying distinct cell clusters based on protein expression and spatial microenvironment.
• Observed specific tissue subregions composed of unique cell clusters and characterized cell-cell interactions, with cells in the same cluster showing association and cells in different clusters avoiding contact.

Conclusions:

• The developed spatial proteomics approach enables high-resolution, multiplexed protein profiling in FFPE tissues.
• The method facilitates the discovery of tissue-specific cell clusters and their spatial organization.
• Provides novel insights into cell-cell interactions within complex biological microenvironments.