Analysis of Protein Cholesterylation by Biorthogonal Labeling

Cholesterol modification (or cholesterylation) is a rare but important posttranslational lipid modification of proteins. So far, two proteins, namely Hedgehog and Smoothened, have been characterized to undergo cholesterylation. It is unknown whether other proteins are similarly modified. Here we present a protocol of a chemical biology method to analyze the Smoothened protein cholesterylation using an azido-conjugated cholesterol analog combined with click chemistry. This method can be applied to analyze Hedgehog cholesterylation, and be extended to identify novel cholesterylated proteins with minor changes.