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 8pmol farnesylated H-Ras in cell lysates. We demonstrate that this method can be used to determine IC(50) 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.
Department of Pharmacy and Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, 1066 EC Amsterdam, The Netherlands.
Natalie M G M
NM
Appels
Hilde
H
Rosing
Trevor C
TC
Stephens
Andrew
A
Hughes
Jan H M
JH
Schellens
Jos H
JH
Beijnen
D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D051231:Farnesyltranstransferase / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D010446:Peptide Fragments / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003903:Deuterium; D051231:Farnesyltranstransferase; D014357:Trypsin; D016283:Proto-Oncogene Proteins p21(ras)