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

Anal Biochem. 2006 May 1;352(1):33-40. doi: 10.1016/j.ab.2006.02.028. Epub 2006 Mar 20.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods*
  • Deuterium
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Farnesyltranstransferase / metabolism
  • Farnesyltranstransferase / pharmacology
  • Humans
  • Inhibitory Concentration 50
  • Models, Biological
  • Peptide Fragments / analysis
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / metabolism
  • Protein Prenylation*
  • Proto-Oncogene Proteins p21(ras) / analysis
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Trypsin*

Substances

  • Enzyme Inhibitors
  • Peptide Fragments
  • Recombinant Proteins
  • Deuterium
  • Farnesyltranstransferase
  • Trypsin
  • Proto-Oncogene Proteins p21(ras)