Quantification of Protein Levels in Single Living Cells

Abstract

Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.

Protein and RNA Quantification of Multiple Genes in Single Cells

“…RPL13a is expressed in all eukaryotic cells at consistent levels, and is routinely used as a reference housekeeping gene for normalization of quantitative mRNA and protein measurements [14,15,16]. The quantification of RPL13a by the insertion of a PQRGFP nols reporter at the end of its coding sequence (Figure 2A) can therefore be used as a measure of a cell's individual transcriptional and translational status [5]. …”

“…For example, we used single-cell protein and mRNA quantification in single cells from human and mouse to determine the covariation between mRNA expression and protein amounts. We found that mRNA expression is a weak predictor of protein translation [5]. Recently, simultaneous measurement of RNA and protein was also performed in single cells using microfluidics, and a high heterogeneity and poor mRNA-to-protein correlations were also observed [9,10,11].…”

“…Large numbers of cells can increase the reliability of results and overcome issues of low sensitivity or the low signal-to-noise ratio of common assays. However, it is becoming increasingly clear that even neighboring cells within tissues or pools of clonal cells are not homogenous [3,4,5,6,7,8]. Cellular dynamics, such as differing transcriptional and translational states, cell signaling, cell cycle, development, and other molecular processes in addition to stochasticity together produce cellular heterogeneity, even in immortalized clonal cell lines.…”

“…We have previously developed a technique that allows for quantitation of protein levels in single cells in vivo [5]. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation.…”

“…The fluorescence intensity is proportional to the number of molecules of the protein of interest produced and is used to determine the relative protein amount within the cell (Figure 1A). Using genome editing tools, PQR constructs can be inserted into any endogenous genomic locus to quantify endogenous protein levels in single living cells [5]. Cells expressing PQR constructs can be noninvasively imaged over time using standard fluorescence microscopy, allowing for other biological assays matched at the single-cell level, such as sequencing, mRNA expression analysis and pharmacological manipulations.…”