Reactive oxygen and nitrogen species, produce dynamic protein modifications and protein complexes in RAW 264.7 macrophage cells.
EMSL Project ID
12291
Abstract
Macrophages play an important role in the immune system by destroying invading pathogens with reactive oxygen and nitrogen radicals capable of modifying macrophage proteins in the process of killing bacteria. Thus we have induced radical generation in macrophages (activation) to look at the effects oxidative stress has on intracellular proteins and protein complexes. We observed an increase and subsequent clearance of nitrated proteins on immunoblot. To validate our findings in whole protein lysate we have introduced a pure nitrated his tagged protein into the system and recovered the protein to spectrally quantify the nitration change. We find a substantial loss of nitrotyrosine in the pure protein post incubation with the lysate proteins. Thus induction of radical generation in macrophages stimulates an increase in clearance of modifications to pure protein. Intact protein mass spectrometry with the 12 tesla FTICR will allow us to pinpoint the nature of the modification clearance, be it reversion from nitrotyrosine to tyrosine, reduction to amino tyrosine or cleavage by cellular degradation machinery. The observed changes in globally nitrated proteins in the macrophage will undoubtedly alter the protein complexes and have implications for the viability of the cell. Thus an in depth characterization of the changes in key complexes associated with oxidative stress will shed light not only on the mechanisms of macrophage activation but also on dynamics of protein nitration within the cell.
Project Details
Project type
Exploratory Research
Start Date
2005-02-24
End Date
2007-06-28
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Lopez-Ferrer D, KK Hixson, HS Smallwood, TC Squier, K Petritis, and RD Smith. 2009. "Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18 O-Labeling Method for Quantitative Proteomics." Analytical Chemistry 81(15):6272-6277.
Smallwood HS, D Lopez-Ferrer, and TC Squier. 2011. "Aging Enhances the Production of Reactive Oxygen Species and Bactericidal Activity in Peritoneal Macrophages by Upregulating Classical Activation Pathways." Biochemistry 50(45):9911-9922. doi:10.1021/bi2011866
Smallwood HS, NM Lourette, CB Boschek, DJ Bigelow, RD Smith, L Pasa-Tolic, and TC Squier. 2007. "Identification of a Denitrase Activity Against Calmodulin in Activated Macrophages Using High-Field Liquid Chromatography - FTICR Mass Spectrometry." Biochemistry 46(37):10498-10505