Chemoproteomic Profiling of Geranyl Pyrophosphate-Binding Proteins

Chemical proteomics has been widely applied in the identification and quantification of targeted proteins. Here we describe a chemoproteomic method, in combination with stable isotope labeling by amino acids in cell culture (SILAC), for the proteome-wide profiling of geranyl pyrophosphate (GPP)-binding proteins. After labeling using a desthiobiotin-GPP acyl phosphate probe, desthiobiotin-conjugated peptides of GPP-binding proteins could be enriched from the tryptic digestion products of complex protein mixtures and subsequently identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To exclude nonspecific binding proteins, we applied SILAC, together with competitive labeling experiments, including high vs. low concentrations of GPP probe, GPP vs. ATP probes, and GPP probe labeling with or without the presence of GPP. Several known or candidate GPP-binding proteins were identified with this method, suggesting the potential application of this method in the study of isoprenoid-interacting proteins and biological functions of isoprenoids.