A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies
| Autor: | Marianne B, Ariganello, Dharani D, Das, Dalibor, Breznan, Christine, MacKinnon-Roy, Fred, Elisma, Aziz, Khanchi, Renaud, Vincent, Prem, Kumarathasan |
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| Rok vydání: | 2018 |
| Předmět: |
Titanium
Proteomics Air Pollutants Proteome Cell Survival Macrophages in vitro exposure toxicoproteomics air pollution Epithelial Cells macrophage Silicon Dioxide air particulate matter lung epithelial cells Mice A549 Cells Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization cytotoxicity Animals Humans Particulate Matter Particle Size Research Articles Research Article mass spectrometry |
| Zdroj: | Journal of Applied Toxicology |
| ISSN: | 1099-1263 |
| Popis: | Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre‐fractionated by molecular weight cut‐off filtration, digested enzymatically and were analyzed by matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre‐fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P To estimate in vitro potencies of ambient air particles we developed an integrated screening platform that enabled protein profiling of cellular responses and cytotoxic analyses. Extensive optimization of the toxicoproteomic analysis was carried out. Cellular proteomic changes were consistent with cytotoxicity findings. The integration of proteomic and cytotoxicity analyses results provided insight into particulate matter cytotoxic reactivity‐related mechanistic information at the molecular level. |
| Databáze: | OpenAIRE |
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