The last years have witnessed an immense improvement in the techniques available for the analysis of the proteome. This is largely due to advances in mass spectrometry (MS) analysis, where electrospray ionization (ESI) and matrixassisted laser desorption ionization (MALDI) represent soft ionization techniques suitable for native and most post-translationally modified peptides (Aebersold and Goodlett, 2001). However, it has been realized that none of the individual methods will be sufficient to provide satisfactory answers to all questions of interest. Each of the proteomic techniques has certain strengths and shortcomings as outlined below. Two-dimensional gel
Handbook of Models for Human Aging
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electrophoresis (2DE) has been and continues to be a widely used technique due to its relatively low cost and maintenance requirements. Continuous improvements are made with regard to reproducibility, solubility (especially of membrane proteins), alternative protein stains, and the differential display of proteins during differential m-gel electrophoresis (DIGE). For DIGE, two separate pools of proteins from cells/tissue of different origin are labeled with two distinct fluorescent dyes, such as 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxy-succinimidyl ester, which can then be individually excited for exclusive monitoring of the labeled proteins from one specific pool (Zhou et al., 2002a). Specific software allows for the three-dimensional representation of the individual protein spots on the gel. Importantly, the fluorescent dyes should be similar in molecular weight and charge so that identical proteins labeled with different dyes comigrate during 2DE. Two labeling strategies for DIGE have been developed, (i) minimal labeling and (ii) saturation labeling (Shaw et al., 2003). During minimal labeling, fluorescent dyes are covalently attached to protein Lys residues. Based on the abundance of Lys residues in proteins, the labeling stoichiometry may exceed more than one molecule of dye per protein. Proteins with multiple dye molecules may migrate differently on 2DE compared to proteins with a single dye or precipitate due to the hydrophobicity of the dyes, enhancing the complexity of samples and rendering the relative quantitation of proteins from different tissues a difficult task. Hence, minimal labeling is run under conditions that ensure that not more than one dye molecule is attached to a single protein. Under these conditions, a significant fraction of proteins remains unlabeled, resulting in an overall loss of sensitivity. The latter may require larger sample volumes for the relative quantitation of low abundance proteins. A new generation of fluorescent dyes has been developed for saturation labeling (Shaw et al., 2003). These dyes are covalently attached to protein Cys residues, which usually show a lower abundance in proteins relative to Lys residues. Saturation labeling is achieved under conditions where each protein Cys residue is labeled. This technique results in a greater sensitivity of the analysis but may still suffer from the precipitation of individual proteins. One drawback of the saturation labeling technique is the potential age-dependent oxidation of protein Cys residues, resulting in the age-dependent loss of labeling sites. In such a scenario the lower stoichiometric labeling of a protein from aged tissue could be misinterpreted as lower expression. The ICAT methodology, described below, suffers from the same problem.
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