Protein expression analysis approaches

Figure 6.15 Applying dual-color ratiometric gene expression labeling approach to protein expression analysis. (From Haab, B.B. etal., Genome Biol., 2, 0004.1-0004.13, 2001. With permission.)...

Kennedy S (2002) The role of proteomics in toxicology identification of biomarkers of toxicity by protein expression analysis. Biomarkers 7 269-290 Li J, Zhang Z, Rosenzweig J, Wang YY, Chan DW (2002) Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer. Clinical Chemistry 48 1296-1304... 

It is therefore clear that until these alternative approaches mature into robust techniques for quantitative protein expression profiling, 2-DE will remain the separation work-horse in many proteomic investigations. This technique has the capacity to support the simultaneous analysis of the changes in expression of hundreds to thousands of proteins and as such it remains unrivalled as an open protein expression profiling approach. 

The ultimate goal of microarray-based expression analysis is to acquire a comprehension of the entire cellular process, in order to exploit and to standardize the multidi-menisional relations between genotype and phenotype. However, an increasingly important parameter, which has not yet been substantially taken into account, is the role of cellular translation. This means that mRNA expression data need to be correlated with the assortment of proteins actually present in the cell. One approach is based on the use of microarrays containing double-stranded DNA probes for the analysis of DNA-protein interaction and, thus, the detection and identification of DNA-binding proteins by means of fluorescence [130] or mass spectrometry analysis [131]. Moreover, substantial efforts are currently under way to develop protein, antibody, or even cell arrays, applicable to the cor-... 

There are two general approaches to cDNA expression, transient expression and stable expression. Transient expression systems are typically based on a viral vector the host cells are infected with cDNA-bearing virus and the cDNA-derived protein is produced. At some point a maximum level of cDNA-derived protein expression is obtained and the protein is harvested for use in incubations. Viral vectors often have cytopathic effects on the host cells which usually precludes analysis of xenobiotic-induced toxicity to the host cell. Stable expression systems can be based on integrating or episomal vectors. With both stable expression approaches, homogeneous, clonally derived populations of cells stably expressing the cDNA are identified and characterized. The properties, advantages and disadvantages of the two approaches are discussed below. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

Quantitation Once protein expression profiling activities characterize qualitative features, the attention turns to delineating protein interactions and mechanistic pathways responsible for disease. These studies also require rapid sequence determination/confirmation combined with accurate and sensitive quantitative analysis. The quantitation approaches would allow for direct comparison of protein amounts (absolute or relative) from a variety of cellular states. Because of the reasons stated previously, quantitative applications are likely to be less dependent on 2-DGE and rely primarily on formats that involve specific purification and/or chromatographic separation with mass spectrometry. 

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