Profiling of C-terminal prenylated proteins using tandem mass tagging

Protein prenylation is a crucial post translational modification that involves the attachment of one or two isoprenoid groups at the C-terminus of a protein, facilitating membrane localization and regulating protein function. Consequently, prenylation is linked to numerous diseases. Identification of prenylated proteins and their quantification is crucial to defining the role of prenylation in these diseases and therapy development. Here, a method for profiling and quantifying prenylated proteins using a bio-orthogonal probe is described. The workflow consists of metabolic incorporation of the probe and click chemistry-mediated biotinylation followed by streptavidin purification and LC-MS³ based proteomic analysis of the enriched proteins. In this chapter, we provide a comprehensive protocol to accomplish this using tandem mass tag (TMT) labels in mammalian cell culture. This includes sample preparation from adherent and suspension cell lines, in-gel fluorescence analysis to verify probe incorporation, click reaction with a biotin handle, streptavidin enrichment of prenylated proteins, multiplexing using TMT labels, LCMS³ data acquisition and data analysis.