Speaker: Thomas P. Conrads, SAIC-Frederick, Frederick, MD 21701 USA

Topic: Proteome Analysis of Camptothecin-Treated Cortical Neurons Using Isotope-Coded Affinity Tags (ICATs)

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Tuesday, January 8, 2002, at 2:00 PM

Abstract: Isotope-coded affinity tags (ICATs) were employed to identify and quantitate changes in protein expression between control and camptothecin-treated mouse cortical neurons. After labeling the proteins extracted from the cortical neuron samples with the light and heavy isotopic versions of the ICAT reagents, the protein extracts were combined, proteolytically digested, and the derivatized peptides isolated using immobilized avidin chromatography. Peptides thus isolated were analyzed by reversed-phase liquid chromatography coupled online to either a conventional ion-trap mass spectrometer (MS) or a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. While a majority of the peptide identifications were accomplished using conventional MS, FTICR was used to quantitate relative abundances of the ICAT-labeled peptides taking advantage of its high resolution, sensitivity, and duty cycle. By using this combination of MS technologies we have thus far identified and quantified the expression of greater than 125 proteins from control and camptothecin-treated mouse cortical neurons. Results from targeted immunohistochemical experiments on protein targets designed using information gained from our ICAT-based MS analyses will also be discussed.