Proteomic and oxPTM analysis of neutrophil extracellular traps

As the most abundant type of white blood cells in mammals, neutrophils form an essential part of the innate immune system. They employ three strategies to eliminate invading microbes: phagocytosis of engulfing bacteria, degranulation involving release of soluble anti-microbial compounds, and a recently discovered mechanism called neutrophil extracellular traps (NETs). NET formation seems to be the consequence of a novel form of active cell death that is neither necrosis nor apoptosis, and they form as part of an innate immune response at sites of acute inflammation. Early work suggested that this cell death program depends on the generation of reactive oxygen species by NADPH oxidase, suggesting the role of a redox-regulated cell signalling pathway. A more recent study has demonstrated the requirement specifically for HOCl production. To investigate further the molecular makeup of NETs and the role of HOCl and signalling mechanisms, we have utilized tandem mass spectrometry techniques to determine the proteins present in NETs and to map the occurrence of oxidative modifications to these proteins. We observed a number of the proteins that have previously been reported as associated with NETs (for example myeloperoxidase, catalase and actin), plus more than one hundred of new proteins that may be associated with NETs including moesin, neuroleukin, annexin and serpin B4. These findings provide a more detailed molecular composition of NETs and a deeper understanding of the role of HOCl-based oxidation in the mechanisms triggering NET release.