کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176636 961867 2006 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Absolute quantification of farnesylated Ras levels in complex samples using liquid chromatography fractionation combined with tryptic digestion and electrospray tandem mass spectrometry
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Absolute quantification of farnesylated Ras levels in complex samples using liquid chromatography fractionation combined with tryptic digestion and electrospray tandem mass spectrometry
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 352, Issue 1, 1 May 2006, Pages 33–40
نویسندگان
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