Abstract Proteins can be modified by lipids in various ways, for example by myristoylation, palmitoylation, farnesylation, and geranylgeranylation—these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analyses of endogenous lipidation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore lipidation sites in HeLa cells, and identified a total of 90 lipidation sites, including 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence lipidated proteins identified by myristic acid analog-based chemical proteomics. Isolation of lipidated peptides from digests prepared with different proteases enabled the identification of different lipidated sites, extending the coverage. Moreover, our peptide-centric approach successfully identified dually modified peptides having myristoylation and palmitoylation. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of various protein lipidations.

Liquid-liquid extraction of lipidated peptides for direct identification of lipidation sites