Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Understanding complex cellular systems necessitates tools for quantifying protein expression within their native context.
• Current methods face limitations in sensitivity, accuracy, and multiplexing capabilities for in situ protein analysis.

Purpose of the Study:

• To develop a highly sensitive and accurate protein in situ profiling approach for multiplexed quantification in single cells.
• To enable the study of protein expression heterogeneity and correlations in various cellular contexts, including FFPE tissues.

Main Methods:

• Utilized off-the-shelf antibodies conjugated with horseradish peroxidase (HRP) and a novel cleavable fluorescent tyramide (CFT).
• Implemented iterative cycles of antibody staining, fluorescence imaging, CFT cleavage, and HRP deactivation.
• Designed and synthesized high-performance CFT with efficient signal erasure (>95%) while preserving epitope integrity.

Main Results:

• Achieved multiplexed protein quantification in single cells with high sensitivity and accuracy.
• Demonstrated efficient (>95%) signal removal by mild chemical reagents, preserving target epitopes.
• Successfully profiled protein expression heterogeneity and correlations in genetically identical cells and FFPE tissues.

Conclusions:

• The developed protein in situ profiling method offers a powerful tool for comprehensive cellular analysis.
• The approach allows for accurate quantification of protein expression levels and their relationships in diverse biological samples.
• This technique enhances the study of cellular composition, function, and regulation through precise protein profiling.