Application of Toxicoproteomics in Profiling Drug Effects
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PURPOSE AND RATIONALE
PROCEDURE
Traditionally, toxicologists define the preliminary risk of a new compound to human safety using animal models supported by histopathological and biochemical approaches. However, despite decades of experience, the extrapolation dilemma and the relevance of animal data to real-life, long-term exposure in humans remained unclear. The genomics revolution of the recent years led to development of many new and innovative technologies that can change this paradigm and address uncertainty issues in the current toxicological practice and safety assessment through the identification of novel key genes, marker proteins, or protein profiles. Thus, these technologies provide a superior alternative to traditional rodent and canine bioassays to identify and accurately assess the safety of chemicals and drug candidates for human safety. Toxicogenomics, the use of DNA microarray for comprehensive RNA expression analysis, has recently caused a great deal of interest (Pennie et al. 2000; Nuwaysir et al. 1999). This technology has been used to monitor changes in gene expression in response to drug treatment. However, analysis of the information produced by toxicogenomics shows some limitation (Anderson and Seilhamer 1997; Mann 1999; Srinivas et al. 2001). Fundamental studies have illustrated the usefulness and potential of the Toxicoproteomics, the proteomic approach, to complement RNA microarray data. Proteomic technology helps identify corresponding changes in the level of protein, which is critical because the protein is the basic component of a cell. Additionally, toxicoproteomics helps resolve issues involving differential protein modifications. These are critical for the function of many proteins, in that they may lead to changes in the activity of gene products. Primarily, the manifestation of protein modifications is the reason for undesired, compound-related effects. Toxicoproteomics helps to determine such changes, and to gain insight into the mode of action of drugs.
Available Technology Platforms
The most common implementation of proteomic analysis involves protein separation two-dimensional gel electrophoresis (2DGE), quantification of proteins with analytical methods for their identification in mass spectrometer (MS), and at the very least data integration and analysis using bioinformatic tools. 2DGE Initially, proteins in a sample are separated according to their isoelectric point in a pH-gradient. Next, the proteins are separated according to size on a SDSpolyacrylamide gel. A dye marker such as coomassie blue, silver or fluorescent dyes then detect the resolved proteins. In order to analyze differentially expressed protein spots in an experimental set of gels a computerized detection and matching system is required. Finally, MS identifies selected protein spots. MS Mass fingerprinting of excised and trypsin-digested gel spots is the method of choice to identify proteins. The masses of the tryptic fragments in a sample are accurately and quickly measured using a matrix assisted lase
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