Isoprenylation is a form of posttranslational modification (PTM), which constitutes the attachment of isoprenoid groups to cysteine of the C-terminus of proteins. Two forms of isoprenylation are distinguished: Three isoprenoid units form the farnesyl group (C₁₅H₂₅) with a mass of 205 Da, whereas the geranylgeranyl group (C₂₀H₃₃) represents four isoprenoid units with a mass of 273 Da.

Prominent examples of isoprenylated proteins are the Ras and Ras-related proteins. Like other small GTPases, these proteins change between two notable conformational stages: the active membrane-associated GTP- and the inactive cytosolic GDP-form. In this context, the isoprenylation serves as a membrane anchor and allows the interaction between isoprenylated Ras and Ras-related proteins with their effector proteins.

All three isoforms of the Ras protein are farnesylated, and the members of the Rho protein superfamily are geranylgeranylated. The Ras-related superfamily of Rab proteins takes an exceptional position: Rab proteins receive two geranylgeranyl moieties from the Rab-geranylgeranyltransferase (Rab-GGTase). After double geranylgeranylation, Rab is integrated into the cell membrane and serves as a molecular switch in intracellular trafficking. Several Rab proteins are aberrantly expressed in tumors¹ but no Rab-specific drugs have been developed yet. Because of the essential isoprenylation for correct localization and function of Rabs, inhibitors of Rab-GGTase have been found². Nonetheless, isoprenylated proteins are low-abundant in complex cell samples. This is why it is extremely difficult to detect them with mass spectrometry, despite sensitive measuring devices. Therefore, enrichment strategies are indispensable.

We have established an elutropic series by using liquid chromatography-mass spectrometry (LC-MS), which discriminates farnesylated, singly and doubly geranylgeranylated peptides. Furthermore, we investigated characteristic gas phase reactions of isoprenylated peptides during ionization and fragmentation.

These results pave the way for future studies on e.g. prenyltransferase inhibitors without further enrichment steps.