A novel approach to tag and identify geranylgeranylated proteins.
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.
Departments of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1489, USA.
Lai N
LN
Chan
Courtenay
C
Hart
Lea
L
Guo
Tamara
T
Nyberg
Brandon S J
BS
Davies
Loren G
LG
Fong
Stephen G
SG
Young
Brian J
BJ
Agnew
Fuyuhiko
F
Tamanoi
D000818:Animals; D001386:Azides / Q000032:analysis* / Q000378:metabolism; D045744:Cell Line, Tumor; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D034904:Lamin Type A / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D017368:Protein Prenylation; D040901:Proteomics / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry; D018631:ras Proteins / Q000032:analysis*
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D001386:Azides; D034904:Lamin Type A; D009687:Nuclear Proteins; D011498:Protein Precursors; D012235:Rhodamines; C090764:prelamin A; C511733:Rap2C protein, human; D018631:ras Proteins