Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes.

Protein prenylation, found in eukaryotes, is a posttranslational modification in which one or two isoprenoid groups are added to the C terminus of selected proteins using either a farnesyl group or a geranylgeranyl group. Prenylation facilitates protein localization mainly to the plasma membrane where the prenylated proteins, including small GTPases, mediate signal transduction pathways. Changes in the level of prenylated proteins may serve a critical function in a variety of diseases. Metabolic labeling using modified isoprenoid probes followed by enrichment and proteomic analysis allows the identities and levels of prenylated proteins to be investigated. In this protocol, we illustrate how the conditions for metabolic labeling are optimized to maximize probe incorporation in HeLa cells through a combination of in-gel fluorescence and densitometric analysis.