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Proteomics deals with the large-scale analysis of proteins expressed by the genome of an organism. In general, the aim is to search for qualitative and quantitative changes in protein expression that occur as a function of development, disease, environmental insults, or treatment. Because of the complexity of the proteome of most organisms, the complete and rapid mapping of protein expression changes and characterization of posttranslational modifications is challenging and will require an analytical effort that exceeds the technology and throughput that exist in most standard biochemistry laboratories (1 While novel automated methods for rapid separation and analysis of proteins are being evaluated in many laboratories, it is widely accepted that twodimensional gel electrophoresis (2-DE) is still the benchmark for large-scale separation of complex protein mixtures. The availability of standard reagents and equipment, and development of many modifications of the 2-DE process, have led to remarkable improvements in reproducibility (2,3). However, it is still difficult to fully duplicate the pattern of protein expression using conventional 2-DE methods (4,5), particularly when conducting multiple comparative and quantitative proteomics experiments in a high-throughput research environment where reproducibility and speed are critically important parameters. [ABSTRACT FROM AUTHOR]

Proteins are composed of different numbers of neutral positively and negatively charged amino acids. Therefore, proteins vary widely in size and have either positive, negative, or zero net charge, depending on the pH of their surroundings. The original two-dimensional gel electrophoresis format was developed almost 30 years ago (1) to exploit this variation in protein charge and size for separation purposes. The isoelectric point of a protein (pI) is the pH at which it has a net zero charge, which, for the majority of proteins, lies between pH 4.0 and 8.0. The sizes of proteins vary widely (10-500 kDa), with an average molecular weight of approx 50 kDa. These two mutually independent properties are exploited by firstly denaturing proteins in urea and then subjecting them to an electric field in a pH gradient established in a low-concentration polyacrylamide gel (originally this pH gradient was formed, using ampholytes, in situ, but now precast immobilized pH gradient [IPG] strips [2-4] are found to be more reliable). In this case, all but the very largest proteins can migrate freely until they reach a pH at which they have no net charge (isoelectric focusing [IEF]). After completion of the focusing, the proteins are denatured in situ, their native charge is saturated with the anionic detergent sodium dodecyl sulphate (SDS), and then the gel is layered, perpendicular to the direction of focusing, on a higher-concentration polyacrylamide gel, and the focused proteins are separated on the basis of size. This gives rise to discrete spots representing one (or perhaps a very small number) of different proteins. [ABSTRACT FROM AUTHOR]

Current research on the molecular basis of human reproductive cancers involves mapping cellular pathways and identifying molecular alterations associated with disease onset and progression. The ability to characterize changes in protein expression that occur during the transition from normal to benign disease to invasive cancer requires homogeneous populations of cells free from contamination by other cell types. With advances in proteomic technologies, proteins can now be quantified in a relatively small number of target cells obtained from a limited amount of tissue. The increased demand for selective isolation of pure cell populations from small quantities of tissue calls for refined protocols for tissue preparation and efficient methods of tissue microdissection that preserve protein integrity. [ABSTRACT FROM AUTHOR]

The importance of serum/plasma as a source of clinically relevant biomarkers/surrogate markers of human disease has increased significantly over the last decade (1,2), and modern proteomic methods have evolved and been adapted to meet the demand. The specific challenges facing serum analysis include the wide dynamic range in the concentration of individual components and the tremendous number of potential variants of glycosylated proteins (3). The most dominant plasma proteins, albumin and immunoglobulin (Ig)G, typically comprise up to 70% of the plasma proteome in abundance. To enable the majority of the remaining, far less abundant proteins to be better visualized by two-dimensional gel electrophoresis (2-DE), these two proteins must first be removed, or at least depleted in relative concentration. There are a number of currently available commercial products from a range of suppliers that enable albumin depletion by chemical affinity, exploiting the remarkable albumin-binding ability of structures closely related to the reactive dye molecule Cibacron blue 3GA (4), and the IgG binding properties of protein G (5). The blue dye has been shown to have a special affinity for proteins containing the dinucleotide fold, a structural feature that is common to several classes of proteins (4). Albumin can be separated from other plasma proteins using lectin affinity, as it is not normally glycosylated, while the majority of classical plasma proteins are. This approach allows both enrichment of lower-abundance proteins, and the study of differences in glycoprotein profiles (6). [ABSTRACT FROM AUTHOR]