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Qualitative and quantitative proteomics workflows with LTQ Orbitrap XL

2008년 3월 19일 09시 45분 06초
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[질량분석기술심포지엄] Recent Advances in MS Instrumentation for Proteomic Perfection: From the Perspective of MS Manufacturing Companies
저자 및
Ryan Bomgarden, Rosa Viner1, Terry Zhang1, Michael Major, Vlad Zabrouskov1
Thermo Fisher Scientific, Rockford, IL,
1Thermo Fisher Scientific, San Jose, CA, USA,
The DNA damage response pathway is critical in maintaining genome stability, and proteins within this pathway are commonly mis-regulated in cancer cells. Camptothecin is an anti-cancer drug that inhibits topoisomerase I DNA unwinding and leads to DNA damage in cells undergoing DNA replication. Here, we employed a mass spectrometry (MS)-based proteomics approach to identify and characterize campothecin-induced DNA damage response proteins in A549 cells over time. Potential signaling proteins were enriched using a PMAC (Phosphoprotein Metal Affinity Chromatography) before MS analysis to selectively capture phosphorylated proteins. Multiplexed iTRAQ labeling was used to determine relative changes in overall protein abundance and phosphorylation state of proteins. The labeled samples were analyzed using nanoLC-ESI-MS/MS with LTQ Orbitrap XL. Selected parent peptide ions were fragmented in the HCD (High Energy Collision Dissociation) collision cell. The HCD MS/MS spectra have high mass accuracy and resolution since they are acquired in the orbitrap and display similar fragmentation pattern to those from a quadrupole collision cell. This analysis resulted in the identification and quantitation of known and novel proteins involved in DNA damage signaling and repair.