Protein separation Proteomics

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

One of the primary challenges facing the field of separation science is the analysis of the entire complement of proteins produced by an organism—a field of research known as proteomics. 2D gel electrophoresis remains the gold standard in protein separations, with the ability to resolve as many as 5000 proteins in a single gel... 

Millea, K.M., Krull, I.S., Cohen, S.A., Gebler, J.C., Berger, S.J. (2006). Integration of multidimensional chromatographic protein separations with a combined top-down and bottom-up proteomic strategy. J. Proteome. Res. 5, 135-146. 

Figure 10.5. Proteomic workflow via protein separation, digestion, MS analysis, and database search.

Proteome analysis in general involves two stages protein separation and subsequent identification and analysis. Multidimensional separations are required in order to result in an adequate resolution of complex protein or peptide... 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

Proteomics is the omics tool that separates and identifies individual proteins from an initial protein mixture. Proteomics is termed global if it identifies proteins from total preparations, or targeted if, for example, a protein superfamily or protein subset is investigated (Blackstock and Weir, 1999 Pandey and Mann, 2000). 

Research on microchip protein analysis has been very active for cellular protein functional assay, clinical diagnostics, and proteomics studies. Once again, the microfluidic technology plays an important role in protein assays. Immunoassay, protein separation, and enzymatic assay will be described in detail in subsequent sections. 

In general, the proteome can be analysed by a broad spectrum of methods. All of them follow similar principles. After treatment of laboratory animals/cells, parallel protein extraction and protein separation is performed. Changes in the proteome are then investigated by differential expression analysis with bioinfor-matics/biostatistics tools before the identification of proteins of interest is done (Anderson and Anderson 1998 Ryan and Patterson 2001 Schrattenholz 2004). The most powerful protein separation technologies... 

The most common implementation of proteomic analysis involves protein separation two-dimensional gel electrophoresis (2DGE), quantification of proteins with analytical methods for their identification in mass spectrometer (MS), and at the very least data integration and analysis using bioinformatic tools. 

All proteomic platforms generally involve separation and identification of proteins. Toxicoproteomics Proteomics applied to toxicology with the aims of identifying critical proteins and pathways affected by chemical and environmental exposures. 

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