Fast and robust proteome screening platform identifies neutrophil extracellular trap formation in the lung in response to cobalt ferrite nanoparticles

ABSTRACT: The goal of this study was to investigate the effects of magnetic iron cobalt oxide nanoparticles (cobalt doped Fe3O4 nanoparticles with increasing amounts of cobalt) after pulmonary exposure while in parallel presenting a proteomics platform that is easily transferable to large-scale nanotoxicology screening as part of an integrated assessment and testing approach for regulation of nanoparticles. Bronchoalveolar lavage fluid (BALF) is a proximal biofluid that can be used to monitor airway inflammation and toxic responses in the lung. It is routinely sampled for differential lung diagnostics and has been discussed as a source for early detection of lung cancer. In order to assess effects of metal oxide nanoparticles upon inhalation, bronchoalveolar lavage fluid from mice dosed by single intratracheal instillation was collected and subjected to classical biocompatibility assays as well as proteome analysis. Magnetic oxide nanoparticles with iron and cobalt oxide (Fe3-xCoxO4) at different ratios (1:0, 3:1, 1:1, 1:3, 0:1) were tested at two concentrations (54 µg, 162 µg per animal) and two time points after instillation (day 1, day 3). As a positive control carbon black nanomaterial known to induce lung inflammation was included for both time points, but only at the high dosage (162 µg per animal). Proteomics experiments were divided into three parts – test of reproducibility, discovery and screening phase. The reproducibility of the newly introduced Evosep One LC system was evaluated by re-measuring of technical replicates (n=16). During the discovery phase, selected representative samples with 3 biological replicates per group (total n=9) including pure iron oxide nanoparticles, pure cobalt oxide nanoparticles and vehicle controls were subjected to in-depth proteome profiling by extensive pre-fractionation and including isobaric tandem mass tag (TMT) labeling following a classical LC-MS/MS setup. This step allowed us to identify affected pathways and generate hypotheses regarding mechanisms of the effects of nanoparticles. During screening, all samples of the study were measured label-free as single-shot injections separated on short gradients of 21 min using the robust, high-throughput Evosep One LC system. This step allowed a fast screening of the 5 different types of magnetic metal oxide nanoparticles on BALF, at two concentrations and two time points together with their representative controls (total n=166). All screening measurements were completed in only 2.7 days.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Bronchoalveolar Lavage

SUBMITTER: Anja Billing  

LAB HEAD: Frank Kjeldsen

PROVIDER: PXD016148 | Pride | 2020-03-26

REPOSITORIES: Pride