A novel approach to tag and identify geranylgeranylated proteins |
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| Authors: | Lai N Chan Courtenay Hart Lea Guo Tamara Nyberg Brandon SJ Davies Loren G Fong Stephen G Young Brian J Agnew Fuyuhiko Tamanoi |
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| Institution: | 1. Departments of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA;2. Molecular Biology Institute, University of California, Los Angeles, CA, USA;3. Molecular Probes‐Invitrogen, Eugene, OR, USA;4. Departments of Medicine and Human Genetics, University of California, Los Angeles, CA, USA |
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| Abstract: | A recently developed proteomic strategy, the “GG‐azide”‐labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido‐geranylgeranyl analog and chemoselective derivatization of azido‐geranylgeranyl‐modified proteins by the “click” chemistry, using a tetramethylrhodamine‐alkyne. The resulting conjugated proteins can be separated by 1‐D or 2‐D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC‐MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG‐azide”‐labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post‐translational modifications. |
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| Keywords: | 2‐D Azide Click chemistry Protein geranylgeranylation Rap2c |
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