Skip to Main Content U.S. Department of Energy
small banner

Proteomics of Membrane Proteins: Relating Calcium Signaling and Oxidative Stress

Diana Bigelow, Principal Investigator

Loss of nitrated proteins in differentiated C2C12 myotubes (mouse muscle cells) after exposure to 250  µM peroxynitrite (PN, a strong antioxidant).
Loss of nitrated proteins in differentiated C2C12 myotubes (mouse muscle cells) after exposure to 250 µM peroxynitrite (PN, a strong antioxidant). Left panel: Myocytes were lysed at various times after treatment and proteins were separated by SDS-PAGE then immunoblotted with anti-nitrotyrosine antibody. Lanes from left to right represent increasing times after PN addition: 0, 10 min, 12 h, 24 h, 36 h, and 72 h. Center panel: The density associated with the extent of nitrotyrosine immunoreactivity plotted as a function of time after PN addition (arrow). Right panel: PN-treated myotubes (10 min after treatment) fixed and immunostained with anti-nitrotyrosine antibody and secondary antibody conjugated with Alexa 488 dye. Nitration is diffuse in the cytoplasm with nucleolar concentration.

A comprehensive understanding of the effects of cellular stress on calcium signaling requires correlated measurements of intracellular calcium transients and global proteomic analysis of calcium-signaling protein complexes and post-translational modifications of proteins. The Proteomics of Membrane Proteins: Relating Calcium Signaling and Oxidative Stress project team at Pacific Northwest National Laboratory (PNNL) is developing technical capabilities for proteomic analyses of membrane protein complexes and post-translational modifications.

Our preliminary work used two biological systems as experimental models: (1) proteins interacting with the voltage-dependent calcium channel from muscle and mouse-brain synaptosomes and (2) skeletal muscle cells exposed to agents that induce oxidative stress to provide an abundant source of oxidatively modified proteins. Our current research is focused on the proteomics of oxidative modifications of myocyte proteins. We are:

  • developing capabilities for the identification of nitrotyrosine-modified proteins in myocytes that may act as cellular sensors of nitrative stress
  • comparing oxidative modifications of skeletal muscle with oxidative modifications of heart muscle during aging
  • determining if the same proteins in tissues are modified as in the cell culture model of oxidative stress
  • determining if these proteins are more highly modified in senescent tissue or may be present in lower abundance due to their degradation.

As part of this project, researchers will produce new methods for identifying nitrotyrosine-modified proteins in cells and tissues to elucidate the biological role of oxidative modification in the cells' response to acute stress as well as to chronic oxidative stress, such as aging. Also, identifying patterns of oxidative modification of signal proteins might provide biomarkers of cellular reactive oxygen species.

Systems Biology at PNNL

Research & Capabilities

Resources

BSI Scientific Areas

Spatial and Temporal Proteomics Projects

Related Projects

Biomolecular Systems Initiative, image