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Fluorescence Visualization of Newly Synthesized Proteins In Mammalian Cells

Julie C. Liu1, Kimberly E. Beatty2, Fang Xie3, Daniela C. Dieterich4, Erin M. Schuman4, Qian Wang3, and David A. Tirrell2. (1) School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, IN 47907, (2) Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, (3) Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter St., Columbia, SC 29208, (4) Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125

Modern proteomic methods enable efficient identification of proteins present in whole cells or in isolated organelles, but a thorough understanding of the proteome requires insight into protein localization as well as protein identity. Previous work demonstrated visualization of newly synthesized proteins in bacterial cells through co-translational introduction of an alkynyl amino acid followed by selective Cu(I)-catalyzed ligation of the alkynyl side chain to the fluorogenic dye 3-azido-7-hydroxycoumarin. In this work, homopropargylglycine (Hpg), a methionine (Met) analog, was used to selectively tag newly synthesized mammalian proteins in a method similar to conventional pulse-chase labeling, and labeled proteins were visualized via fluorescence. Pulses as short as 15 minutes with no chase provided a five-fold fluorescent enhancement compared to controls. We demonstrate that the extent of labeling can be controlled through the ratio of Hpg to Met and the concentration of the copper(I) catalyst. This fluorescent-labeling method worked on a wide variety of cell types, and, in many cases, the most intense fluorescence appeared to localize to nucleolar structures. This study thus demonstrates a facile method for visualizing proteins based on the timing of protein synthesis even when the sequence, structure, or function of the proteins is unknown.