Protein detection and quantitation technologies for gel-based proteome analysis.

Abstract

Numerous protein detection and quantitation methods for gel-based proteomics have been devised that can be classified in three major categories: (1) Universal (or "general") detection techniques, which include staining with anionic dyes (e.g., Coomassie brilliant blue), reverse (or "negative") staining with metal cations (e.g., imidazole-zinc), silver staining, fluorescent staining or labeling, and radiolabeling, (2) specific staining methods for the detection of post-translational modifications (e.g., glycosylation or phosphorylation), and (3) differential display techniques for the separation of multiple, covalently tagged samples in a single two-dimensional electrophoresis (2-DE) gel, followed by consecutive and independent visualization of these proteins to minimize methodical variations in spot positions and in protein abundance, to simplify image analysis, as well as to improve protein quantitation by including an internal standard. The most important properties of protein detection methods applied in proteome analysis include high sensitivity (i.e., low detection limit), wide linear dynamic range for quantitative accuracy, reproducibility, cost-efficiency, ease of use, and compatibility with downstream protein identification or characterization technologies, such as mass spectrometry (MS). Regrettably, no single detection method meets all these requirements, albeit fluorescence-based technologies are currently favored for most applications; hence, the major focus of this chapter is on fluorescent-dye-based protein detection and quantitation techniques. Although satisfying results with respect to sensitivity and reproducibility are also obtained by methods based on radioactive labeling of proteins (which is still unsurpassed in terms of sensitivity), radiolabeling is, however, largely impractical for routine proteomic profiling because of the costs and the health and safety concerns associated with handling radioactive compounds.