Absolute quantification of farnesylated Ras levels in complex samples using liquid chromatography fractionation combined with tryptic digestion and electrospray tandem mass spectrometry

Farnesylation is the first posttranslational modification of H-Ras proteins, which can be blocked by farnesyl transferase inhibitors. We developed a sensitive and quantitative bioanalytical assay to determine the absolute amounts of farnesylated H-Ras in tumor cell lysates before and after administration of these compounds. Farnesylated H-Ras was isolated with reversed-phase liquid chromatography. Subsequently, the isolated fraction was digested with trypsin and analyzed with electrospray–tandem mass spectrometry. The farnesylated peptide consisting of cysteine-valine-leucine-serine (f-CVLS) proved to be a suitable signature peptide. Its deuterated analogue was used as internal standard for absolute quantification. With this method, we obtained a lower limit of quantification of 8 pmol farnesylated H-Ras in cell lysates. We demonstrate that this method can be used to determine IC50 values of farnesyl transferase inhibitors through absolute quantification of the amount of f-CVLS present after incubation with different concentrations of a farnesyl transferase inhibitor. The signature peptide approach combined with prefractionation may be used as a pharmacodynamic assay to determine dose–effect relationships of farnesyl transferase inhibitors in (pre)clinical studies.