Proteomics of model organisms (A. thaliana and S. cerevisiae) exposed to nanoparticles.

Abstract

Engineered nanomaterials (ENMs) are structures with size in the range of 1-100 nm, and characterized by properties due to their small size and surface reactivity that make them suitable for many industrial applications. Nanotechnology is a rapidly growing industry producing tons of ENMs each year. Therefore, due to the wide diffusion of ENMs and of the lack of information about their mechanisms of their interaction with living organisms, it is crucial to assess the risks linked to their diffusion in the environment. In particular, Cadmium sulfide quantum dots (CdS QDs) are widely used in the electronic industry to produce semiconductors, LED, optical devices, solar energy cells, and medical devices. The aim of the research carried out during the Ph.D. thesis was to evaluate the response of model systems to CdS QDs, exploiting proteomics approaches. The first part of this work was performed on the model plant Arabidopsis thaliana L. (Heynh). In a previous study a collection of 398 mutants of A. thaliana were utilized for selection of mutants resistant mutants to normally toxic concentrations of CdS QDs. Two of the selected mutants (atnp01 and atnp02) were characterized phenotypically and the genotypically, demonstrating that the mechanism of resistance to CdS QDs was minimally overlapped with resistance to Cd ions, supporting the possibility that nanoparticles have specific toxicity mechanisms. In this work, the proteomic analysis was performed on crude protein extracts, obtained from whole seedlings of atnp01, atnp02, and wild type, grown on agarized MS, treated with 80 mg/L CdS QDs and non-treated. The plants whole proteome was separated by 2D gel electrophoresis, analyzed utilizing PDQuest software, and proteins differentially abundant between wild-type and each of the mutants were marked. Nighty-eight (98) proteins, whose abundance was statistically (Student's t test p<0,05) different in response to the experimental conditions, were identified by MALDI-TOF/MS and searched within MASCOT and UNIPROT database to infer their possible role in the plant response to nanoparticles, in particular in the resistance to CdS QDs. Gene Ontology analysis of the identified proteins revealed that the main categories modulated by CdS QDs treatment were: oxidative stress response, protein ubiquitination and degradation, energy and sugar metabolism. The aim of the second part of the work was to assess the response of the model system Saccharomyces cerevisiae to CdS QDs, developing and comparing two different protein isolation methods, set respectively on gel-based and gel-free proteomics. A comparative study on the two quantitative methods frequently used in proteomics, 2-DE (dimensional gel electrophoresis) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. The first method is a familiar techniques used in gel based quantitative proteomics, the second method is a new LC (liquid chromatography) -based technique which is gradually gaining scientific consent. The iTRAQ method allows for simultaneous protein identification and quantification (relative and absolute) obtained at the MS/MS level from peptide fragments (m/z range 150–2200) and low mass reporter ions, respectively. iTRAQ is conceptually smart, since peptides are labelled at the N terminus and at the ε side chain of lysines. Thus, every peptide ion selected for fragmentation generates a sequence with abundant data for proteins up to eight samples, thanks to multiplex reagents design. The proteomic analysis was performed in yeast cells collected in the exponential phases of growth in liquid YPD (yeast extract peptone dextrose), without any supplementation, or with a supplementation of: 0.25 mg L−1 nystatin, 100 mg L−1 CdS QDs and 0.25 mg L−1 nystatin plus 100 mg L−1 CdS QDS. This comparative analysis was used to identify differences in proteins abundance in the controls with respect to the treatments. The data were analyzed through different bioinformatics tools to identify the proteins and the main pathways of response to CdS QDs. Pathway analysis of the identified proteins revealed that the main classes modulated by CdS QDs treatment were: glycolysis and gluconeogenesis, ribosome, protein processing in endoplasmic reticulum (ER), biosynthesis of secondary metabolism and biosynthesis of amino acids. The results obtained could provide information regarding mechanisms, biological process, and genes involved in response to nanomaterials in yeast, which could be extended to superior eukaryotes.