Anne-Cécile Massart

Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS

Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid-liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins.

Effect-based proteomic detection of growth promoter abuse

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17β-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.