The proteomics work package for Predtox project consists of 2D-DIGE, 2D-PAGE and SELDI experiments to find modulated proteins in target organs (liver, kidney), plasma and urine.
2D-DIGE experiments are performed on proteins from liver and/or kidney for a subset of 10 in vivo studies. Relative quantification of 2D-DIGE spots is done by image analysis and modulated proteins are identified by MALDI MS peptide mass fingerprinting in combination with peptide fragment analysis by MALDI TOF/TOF tandem MS.
2D-PAGE experiments are carried out on urinary proteins for a subset of 10 in vivo studies. Quantification is carried out by semi-quantitative mass spectrometry-based methods. Proteins are identified by MALDI MS peptide mass fingerprinting.
SELDI measurements are done on proteins from plasma samples, liver or kidney from 16 in vivo studies on two pre-selected chromatographic surfaces. Differential expression analysis and identification of potential biomarkers are done using SELDI software. Up to 12 differentially expressed masses from plasma will be identified by a combination of chromatography and mass spectrometry-based methods, and validated by Western blots.
As several laboratories participate in the 2D-DIGE and SELDI experiments, pilot studies were carried out to ensure technical harmonisation between sites. All the results from the proteomics work package are submitted to the Predtox database in the appropriate format for further analysis in combination with the other classical and ‘omics approaches.