A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.
A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.
Kho
Yoonjung
Y
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.
Kim
Sung Chan
SC
Jiang
Chen
C
Barma
Deb
D
Kwon
Sung Won
SW
Cheng
Jinke
J
Jaunbergs
Janis
J
Weinbaum
Carolyn
C
Tamanoi
Fuyuhiko
F
Falck
John
J
Zhao
Yingming
Y
D000818:Animals; D001386:Azides / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D015394:Molecular Structure; D017368:Protein Prenylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D013194:Staining and Labeling / Q000379:methods*; D018631:ras Proteins / Q000378:metabolism
D001386:Azides; D011506:Proteins; D018631:ras Proteins