Title: Label-free mass spectrometry-based vs 2D gel-based quantitative approach to identify the proteomic changes that occur in Caco-2 cells exposed to gold nanoparticles (AuNPs)
Publisher: Italian Proteomics Association
Publication Year: 2015
JRC N°: JRC96657
ISBN: 978-88-7959-877-4
URI: http://publications.jrc.ec.europa.eu/repository/handle/JRC96657
Type: Articles in periodicals and books
Abstract: BACKGROUND-AIM Emerging approaches in the area of human exposure to nanomaterials and potential effects combine the use of in vitro systems and post-genomics techniques. In this work, we investigate the modification that occurs in the proteome profile of the human colon adenocarcinoma (Caco-2) cell line when exposed to 5 and 30 nm gold nanoparticles (AuNPs). On one hand, in gel protein digestion protocols offer a simple way of protein pre-fractionation through two-dimensional gel electrophoresis (2DE). On the other hand, label-free mass spectrometry (MS)-based proteomics facilitates the throughput of novel proteomic approaches. Two distinct protocols were used to process identical proteome sample from Caco-2 cells. METHODS The gel-based proteomics approach combines protein separation/quantitation by 2DE followed by in gel digestion and identification of de-regulated proteins by MS. Proteome Discoverer with the Sequest search algorithm was used in combination with UniProtKB. The label-free MS approach is based on the quantitative liquid chromatography – high-resolution mass spectrometry LTQ Orbitrap XL platform. Data were analysed by means of Progenesis QI for Proteomics. In both protocols, data from 6 independent biological replicates were combined. RESULTS Using 2DE and in gel digestion method, a total number of 66 proteins were found significant differentially expressed between the two groups (5 and 30 nm AuNPs). The in solution digest protocol conducted in combination with label-free mass spectrometry gave good performances in terms of number of detected proteins (>100). By investigating the differentially expressed proteins triggered by AuNPs exposure, it was possible to identify several pathways using Ingenuity Pathway Analysis (stress response, small molecule biochemistry, cellular assembly and organization, cellular growth and proliferation). CONCLUSIONS The present study integrates two different proteomics approaches to elucidate the toxicological effects at cellular level of two different sizes of AuNPs on a Caco-2 cell model. We believe that nowadays progresses in proteomics technologies offer great advantages in terms of sensitivity and specificity and their combination represents, compared to traditional study, a cornerstone to investigate effects of external stimuli on a living system.
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