Development of a Phosphoproteomics Approach to Study GPCR-Mediated Signaling Events in a Human Prostate Carcinoma Cell Line

摘要

We successfully demonstrated that our phosphoproteomics strategy employing SCX and TiO_(2) affinity chromatography combined with nano-HPLC/ESI-MS/MS analyses employing CID and ETD fragmentation is well suited to identify phosphoproteins and phosphorylation sites in proteins from orthovanadate-treated LNCaP cells. We were able to detect 73 phosphorylation sites in 67 proteins. ETD spectra yield generally lower Mascot peptide scores than CID spectra, presumably due to the lower intensities of ETD fragment ions compared to the intensity of the remaining precursor ion. We will next apply this strategy to the study of PSGR-mediated signaling events following stimulation of LNCaP cells with steroid derivatives. In our continuative work we will combine this phosphoprotoemics approach with stable isotope labeling in order to obtain time-resolved data on PSGR-mediated siganling networks.