(689b) Development of a Fluorescent Barcoding System for High-Throughput, Single-Cell Analysis

Single-cell analysis of protein expression enables the understanding of heterogenous proteome responses that are often masked at the population-level. We have developed a fluorescent barcoding system that allows for the high-throughput analysis of cell-based proteomic libraries, such as the yeast GFP fusion collection and the yeast knockout collection, using flow cytometry. Fluorescent barcodes are composed of epitope tags, present in different combinations and copy number, that produce unique fluorescent signatures when labeled with fluorophore-conjugated anti-epitope tag antibodies. Up to five distinct fluorescence intensities were generated per epitope by varying copy number, enabling the potential creation of over 600 unique fluorescent barcodes using seven-color flow cytometry. We assigned distinct, genetically encoded fluorescent barcodes to members of the yeast-GFP fusion library, such that each clone constitutively expresses a unique fluorescent barcode on its surface and a GFP fusion protein. Distinct barcode fluorescent signatures and GFP-fusion protein expression changes were simultaneously measured with single-cell resolution using flow cytometry. We have developed a program to mathematically deconvolve mixed barcode population data using a Gaussian mixture modeling approach. We are currently applying the barcoding system to study the dynamic proteome response to oxidative stress in order to understand the role of oxidative stress in disease and potentially elucidate new therapeutic targets.