期刊
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
卷 18, 期 22, 页码 5995-5999出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmcl.2008.08.046
关键词
Azide; Alkyne; Proteomics; Fluorescence; Click chemistry; Microscopy
资金
- NIH [GM62523]
- ARO-sponsored Institute for Collaborative Biotechnologies
- Joseph J. Jacobs Institute for Molecular Engineering for Medicine
- Hertz Foundation
- PEO
- AAAS (Alan E. Leviton Award)
The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission three-to sevenfold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells. (C) 2008 Published by Elsevier Ltd.
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