Quantification of Protein Levels in Single Living Cells.

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Accurate measurement of protein levels is crucial for understanding fundamental molecular processes within cells.
• Existing methods may have limitations in quantifying protein expression at the single-cell level in vivo.
• Investigating gene-to-phenotype relationships requires precise tools for protein quantitation.

Purpose of the Study:

• To develop and validate a novel technique for accurate, in vivo quantitation of protein levels in single cells.
• To establish a method that allows for stoichiometric measurement of protein expression using fluorescent reporters.
• To enable more precise analysis of gene expression and its downstream effects.

Main Methods:

• Development of the protein quantitation ratioing (PQR) technique, utilizing a genetic tag for stoichiometric expression of a fluorescent reporter and the protein of interest.
• Application of PQR for quantifying protein expression of various genes through quantitative imaging, electrophysiology, and phenotypic analysis.
• Genome editing was employed to integrate Protein Quantitation Reporters into endogenous genomic loci across three different species.

Main Results:

• The PQR technique enables fluorescence intensity to be directly proportional to the number of protein molecules produced.
• Demonstrated successful quantitation of endogenous protein levels using PQR in multiple genomes.
• Validated the accuracy of PQR in measuring relative protein amounts within single cells.

Conclusions:

• The PQR technique offers a significant advancement for precise protein quantitation in single cells.
• This method facilitates more accurate investigations into gene-to-phenotype relationships.
• PQR provides a versatile tool for a broad spectrum of quantitative biological experiments.