A matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry‐based toxicoproteomic screening method to assess in vitro particle potencies

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 (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 < 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.

In vitro toxicity screening of amorphous silica nanoparticles using mitochondrial fraction exposure followed by MS-based proteomic analysis

“…Filtrates obtained from MWCO fractionation were evaporated to complete dryness under a gentle stream of high-purity N 2 (g). These evaporated fractions were resuspended in 25 μL of 50 mM NH 4 OAc (pH 7.4) and were treated with 2 μL ProteaseMAX and 10 μL of Trypsin/Lys C enzyme solution (50 μg mL −1 ) for enzymatic digestion, 26 vortexed gently and then centrifuged at 5000 g (1 min). Samples were then sonicated for 5 min in an ice bath, centrifuged at 5000 g (1 min), and incubated overnight in a water bath, at 37 °C.…”

“…Survey MS scans for peptide m / z profiles were obtained and mass spectral data were analysed using the bioinformatics software ClinPro Tools (Bruker Daltonics Ltd, Bremen, Germany) to assess SiNP exposure-related mitochondrial protein profile changes. 25,26…”

“…Survey MS scans for peptide m/z profiles were obtained and mass spectral data were analysed using the bioinformatics software ClinPro Tools (Bruker Daltonics Ltd, Bremen, Germany) to assess SiNP exposure-related mitochondrial protein profile changes. 25,26 LC-MS/MS analysis and protein identification Another set of enzymatically digested samples were desalted using ZipTip C 18 pipette tips (Millipore, catalog number ZTC18S096), according to the manufacturer's protocol, were dried by evaporation using a Speed Vac concentrator (Savant) and were stored at −20 °C prior to MS analysis. Before MS analysis, the dried peptides were immediately re-suspended in 1% FA.…”