Fig. 1.

Strategy for direct identification of lipidated peptides.A, comparison of the numbers of lipidated peptides identified by different methods. The numbers of identified myristoylated or palmitoylated peptides from HeLa cell protein digests spiked with synthetic lipidated peptides were compared with or without LLE using ethyl acetate and a conventional or high-ACN gradient. In the experiments with LLE used 25 μg peptides, while in the experiments without LLE, the amount of peptides injected was 250 ng or 500 ng, which is empirically considered to be approximately equal to the LLE-isolated peptides from 25 μg input. The spiked synthetic peptides are shown in supplemental Fig. S1. Conventional gradient: 4 to 36% acetonitrile (ACN) in 30 min, 36 to 80% ACN in 3 min, 80% ACN for 10 min. High-ACN gradient: 30 to 56% ACN in 30 min, 56 to 80% ACN in 3 min, 80% ACN for 10 min. B, scheme of extraction and analysis of lipidated peptides. Protein digests are prepared according to a conventional shotgun proteomics protocol. Hydrophobic lipidated peptides are partitioned into organic solvents and isolated by liquid-liquid extraction. Isolated peptides are analyzed by nanoLC/MS/MS, focusing on the high ACN concentration range.