The aim of global proteomics studies is to examine all of the proteins in a cell or tissue simultaneously. For example, a typical global analysis might involve the quantitative comparison of the protein profiles in normal cells and diseased cells, or comparison of a cell line before and after exposure to a drug. The goal is to identify specific proteins connected with the disorder or drug treatment. Such investigations may result in the discovery of new drug targets and diagnostic markers, provide mechanistic insights, or define critical signaling pathways. For example, protein expression profiling has resulted in the annotation of some of the proteins that are differentially regulated in normal human luminal and myoepithelial breast cells (Page et al., 1999). Such studies are also contributing to the compilation of databases for the study of bladder cancer (Celis et al., 1999) and the human prostate (Nelson et al., 2000), while a number of potential cancer biomarkers have been identified by similar means (Srinivas et al., 2001). Although the value of successfully elucidating cellular and pathological processes on a proteome-wide level is indisputable, the scale of the undertaking is not trivial and becomes more challenging as the complexity of the organism increases, as discussed above. Thus, the ability to effectively analyze proteins on a global scale is subject to the capacities of proteomic technologies for the resolution of a large number of proteins with distinct properties, as well as their detection over a wide range of concentrations. There are multiple alternative methods for enhancing the comprehensiveness of protein profile analyses of complex proteins, including prefractionation of the pro-teome by various methods, followed by analysis of all fractions.
Another strategy for addressing the greater complexity of most proteomes relative to currently available separation and analysis methods is to use targeted proteomics. This approach focuses on a well defined subset of the pro-teome that can be reproducibly isolated. Reduced complexity is an inherent advantage of this approach. The closer match between protein complexity of many subproteomes and available analytical tools suggests that it may be more efficient and feasible to combine the results of multiple sub-proteomic investigations to define complete proteomes than to employ a direct global proteome analysis approach.
Examples of sub-proteomes that are being studied include large macromolecular complexes and cellular machines, specific classes of proteins, and organelles (see Table 22.1.1). Although the same analytical technologies may usually be employed for global and targeted proteomic studies, the latter studies require specific initial strategies to isolate the appropriate sub-proteome components. Frequently, multiple alternative methods are available for isolating particular subsets of the proteome. For example, phosphoproteins can be isolated using antibodies against phosphoamino acids
Gel-Based Proteome Analysis
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