Protein complexes altered by oxidative stress and determined by FTICR-MS
EMSL Project ID
25891
Abstract
To clarify the dynamics of radical mediated modifications, originating from reactive oxygen species (ROS) produced by macrophages, we analyzed modifications to calmodulin (CaM) using immunoblotting and capillary reversed phase liquid chromatography (RPLC) coupled with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. RPLC-FTICR revealed a dramatic reduction in the abundance and diversity of modified species post incubation with macrophage proteins. Specifically, we show that macrophage repair pathways can revert nitrated tyrosines and oxidized methionines on CaM to their original unmodified state. In addition, we found that macrophage proteins selectively cleave the C-terminal lysine from oxidatively modified CaM, which is likely to alter CaM function. Furthermore, we propose that the cleaved CaM makes a useful biomarker of intracellular oxidative stress conditions due to both the oxidation dependence of this lysine cleavage and the stability of the cleavage product, contrary to nitrotyrosine and methionine sulfoxide, which we clearly demonstrated to be transient reversible modifications. Currently we propose to do an in depth characterization of the changes in oxidative modifications to proteins and clarify how the modifications affect protein-protein interactions which will greatly further our understanding of normal cellular processes. The specific experimental goals of this proposal are: (1) to determine if there is a recognition requirements for repair of modified protein (i.e. CaM); (2) determine how oxidative PTMs to proteins alter protein-protein interactions using CaM as a model system. This project will particularly benefit from the EMSL Resource capabilities; in particular, high resolution separations, high mass measurement accuracy, and the ability to characterize intact proteins will enable identification of post-translationally modified proteins with high confidence and sensitivity.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-06-05
End Date
2009-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages.
Smallwood HS, Lopez-Ferrer D, Eberlein PE, Watson DJ, Squier TC.
Chem Res Toxicol. 2009 Feb 5. [Epub ahead of print]