Integrated top-down and bottom-up proteomics strategy to identify multisite and multistep modifications on radiation sensitive proteins
EMSL Project ID
30441
Abstract
In order for a cell to mount the appropriate response to a stimulus such as radiation, it must have the necessary biosensors capable of assessing inputs from multiple intracellular and extracellular signaling pathways. These multiple signaling components will lead to post-translational modifications (PTMs) on existing proteins that initiate phenotypic responses. Phosphorylation, one of the most important and best characterized post-translational modifications, is essential in signal transduction, gene regulation, and metabolic control in cells, especially in response to intracellular and extracellular changes and stimuli. We hypothesize that many proteins are regulated by specific combinations of modifications that ultimately control their activity in response to stimuli such as ionizing radiation. There is increasing evidence to support this hypothesis for many proteins including histones and p53 but we currently have a very limited knowledge about the relevant protein isoforms associated with specific cellular responses. Using a bottom up proteomics strategy, we have identified hundreds of radiation sensitive phosphorylation sites and many proteins that contain multiple phosphorylations showing differential radiation dose responsiveness (Yang et al, 2006; Yang and Stenoien, in preparation). It is unclear however, whether these multiple phosphorylations occur on the same protein molecule and if specific combinations of modifications (both multiple phosphorylations and phosphorylations in combination with other PTMs) are associated with specific cellular responses such as DNA repair and apoptosis. To assess the relevance of multisite modifications in controlling the cellular response to ionizing radiation, we will utilize a new EMSL capability for intact protein analysis using high performance liquid chromatography (LC) and mass spectrometry (MS) which has the capability to identify multiple modifications including phosphorylation on intact proteins. We will apply this technology platform to identify multisite modifications on protein complexes composed of histones and p53 isolated from radiation treated cells.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-08-21
End Date
2011-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Hu Z, YM Kim, MB Sowa, RJ Robinson, X Gao, TO Metz, WF Morgan, and Q Zhang. 2012. "Metabolomic Response of Human Skin Tissue to Low Dose Ionizing Radiation." Molecular Biosystems. doi:10.1039/c2mb2506if
Tian Z, R Zhao, N Tolic, RJ Moore, DL Stenoien, EW Robinson, RD Smith, and L Pasa-Tolic. 2010. "Two-dimensional liquid chromatography system for online top-down mass spectrometry." Proteomics 10(20):3610-3620. doi:10.1002/pmic.201000367