Liquid chromatography\u2013tandem mass spectrometry and passive sampling: powerful tools for the determination of emerging pollutants in water for human consumption

Among the wide range of emerging pollutants, perfluorinated compounds and various pharmaceuticals, such as nonsteroidal anti-inflammatory drugs, are showing growing concern. These contaminants can be found in freshwater ecosystems because of their incomplete removal during wastewater treatments so, their water solubility and poor degradability result in their continuous discharge and pseudo-persistent contamination. Usually, expected levels of these analytes are particularly low; therefore, sensitive and selective analytical techniques are required for their determination. Moreover, sampling and preconcentration are fundamental steps to reach the low detection limits required. The polar organic chemical integrative sampler (POCIS) represents a modern sampling approach that allows the in-situ preconcentration of ultra-trace pollutants. In this work, a fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of diclofenac, ketoprofen, mefenamic acid, naproxen, ibuprofen, perfluorooctanoic acid, perfluorooctanesulfonate and caffeine in water for human consumption. The chromatographic separation of analytes was achieved in less than 6 min. Quantitative analysis was performed in multiple reaction monitoring mode using ketoprofen-d3 as internal standard. Two different sites of Northern Italy were studied deploying POCIS for four weeks in both inlet and outlet of two drinking water treatment plants. The evaluation of time-weighted average concentration of contaminants was accomplished after the calibration of POCIS; to this aim, the sampling rate values for each compound were obtained by means of a simple calibration system developed in our laboratory. Ketoprofen, perfluorooctane sulfonate, perfluorooctanoate and caffeine were measured in both sites at the ng l 121 level. Copyright \ua9 2016 John Wiley & Sons, Ltd

Proteomic profiling and post-translational modifications in human keratinocytes treated with Mucuna pruriens leaf extract

Mucuna pruriens (Mp) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure. MATERIAL AND METHODS: The effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS). RESULTS: Eighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9. CONCLUSIONS: Our findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs)

Proteomic analysis of 4-hydroxynonenal and nitrotyrosine modified proteins in RTT fibroblasts

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)—a major secondary product of lipid peroxidation— and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients

(A) Measurement of NAD⁺ levels in HEK293 transfected cells.

<p>Cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5, 2, and 24 h. The concentration of NAD⁺ was determined using 2×10⁵ transfected cells. Error bars represent the standard deviation from two independent experiments. (**p<0.01; *p<0.05). (<b>B</b>) <b>DSBs repair analysis evaluated by γ-H2AX foci.</b> HEK293 cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5, 2, and 24 h. Fixed cells were stained with anti-γ-H2AX, and the number of γ-H2AX foci was counted in 50 cells/experiment, in two repeated experiments. (**p<0.01; *p<0.05). (C) Co-immunoprecipitation experiments aimed at evaluating the interaction of MRE11 with the Strep-tagged proteins and PARP1. HEK293 cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5 h. Protein eluates were immunoprecipitated with MRE11 and blotted with anti-Strep-tag and anti-PARP1 mouse monoclonal antibodies.</p

Empty vector (false positive).

<p>Empty vector (false positive).</p

Novel interactors of NBN, p26 and p70 fragments.

<p>Novel interactors of NBN, p26 and p70 fragments.</p

p70 fragment interactors.

<p>p70 fragment interactors.</p

Interactome of the p70 fragment.

<p>The interactome was gleaned by merging the results obtained through SDS-PAGE-MS/MS analysis of the streptavidin chromatography eluates obtained from cells expressing the p70 protein fragment (<b>A</b>) in control conditions or (<b>B</b>) following 2 Gy of X-rays. For details, see the text.</p