1. A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.
- Author
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Ariganello MB, Das DD, Breznan D, MacKinnon-Roy C, Elisma F, Khanchi A, Vincent R, and Kumarathasan P
- Subjects
- A549 Cells, Animals, Cell Survival drug effects, Epithelial Cells metabolism, Humans, Macrophages metabolism, Mice, Particle Size, Proteomics methods, Silicon Dioxide toxicity, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Titanium toxicity, Air Pollutants toxicity, Epithelial Cells drug effects, Macrophages drug effects, Particulate Matter toxicity, Proteome metabolism
- Abstract
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 (TiO
2 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 < 0.05) PM dose-dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency-based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies., (Copyright © 2018 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.)- Published
- 2018
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