Fast and Robust Proteome Screening Platform Identifies Neutrophil Extracellular Trap Formation in the Lung in Response to Cobalt Ferrite Nanoparticles.

Despite broad application of magnetic nanoparticles in biomedicine and electronics, only a few studies on biocompatibility are available. In this study, toxicity of magnetic metal oxide nanoparticles on the respiratory system was examined by single intratracheal instillation in mice. Bronchoalveolar lavage fluid (BALF) samples were collected for proteome analyses by LC-MS/MS, testing FeO nanoparticles doped with increasing amounts of cobalt (FeO, CoFeO with an iron to cobalt ratio 5:1, 3:1, 1:3, CoO) at two doses (54 μg, 162 μg per animal) and two time points (day 1 and 3 days postinstillation). In discovery phase, in-depth proteome profiling of a few representative samples allowed for comprehensive pathway analyses. Clustering of the 681 differentially expressed proteins (FDR < 0.05) revealed general as well as metal oxide specific responses with an overall strong induction of innate immunity and activation of the complement system. The highest expression increase could be found for a cluster of 39 proteins, which displayed strong dose-dependency to iron oxide and can be attributed to neutrophil extracellular trap (NET) formation. In-depth proteome analysis expanded the knowledge of NET formation. During screening, all BALF samples of the study ( = 166) were measured label-free as single-injections after a short gradient (21 min) LC separation using the Evosep One system, validating the findings from the discovery and defining protein signatures which enable discrimination of lung inflammation. We demonstrate a proteomics-based toxicity screening with high sample throughput easily transferrable to other nanoparticle types. Data are available ProteomeXchange with identifier PXD016148.