Proteomics

Proteomics, a term coined in 1995, aims to obtain a global and integrated view of the biology of cells by studying all the proteins of a cell rather than each one individually (Wasinger et al. 1995; Wilkins et al. 1995). As stated in Sect. 1, there are many more proteins than genes in cells because of post-transcriptional modifications. This implies that the gene characterisation is not sufficient to assess the types and functions of proteins in the organism.

Complete draft sequences are now available for the genomes of many eubacteria, several archaebacteria, several unicellular eukaryotes, several plants and animals. As these sequences have accumulated it has become increasingly apparent that new methods are needed to exploit the information that they contain. Three types of proteomics have been applied in biology (Graves and Haystead 2002). Protein expression proteomics concerns the quantitative study of protein expression between samples that differ by some variables. This approach has allowed the determination of specific proteins in signal transduction or in disease processes. Structural proteomics aims to identify all proteins of a protein complex (e.g. in subcellular organelles) assessing their location and characterising all protein-protein interactions. Functionalproteomics,abroad termformany specifically directed proteomic approaches, allows the study and characterisation of a selected group of proteins and can provide important information about protein signalling, disease mechanisms or protein-drug interactions. Thus, the proteomics approach can concern either the entire proteome or subproteomes.

In any approach, the analytical method should resolve the protein mixture into its individual components so that each protein can be identified and characterised. The predominant method used for the separation of proteins is two-dimensional (2-DE) polyacrylamide gel electrophoresis where proteins are separated by two distinct properties: the net charge in the first dimension and the molecular mass in the second dimension (Graves and Haystead 2002). The immobilisation of a pH gradient on the gel has increased the resolution and reproducibility of the technique (Bjellqvist et al. 1993; Gorg et al. 2000). In particular, 2-DE has the ability to resolve proteins that have been subjected to post-translational modifications, because these protein modifications confer a change in protein mass and charge (Graves and Haystead 2002). The main application of the technique is the comparison between two samples so as to determine qualitative and quantitative differences concerning the protein expression. Indeed, the appearance or disappearance of spots can provide information about differential protein expression, while the intensity of the spots can give quantitative information about protein expression levels (Graves and Haystead 2002). A recent advance in 2-DE is represented by difference gel electrophoresis (DIGE) where two proteins fluorescently tagged with two different dyes are run on the same 2-D gel (Unlu et al. 1997). After the run, fluorescence imaging of the gel is used to create two images which are superimposed to identify pattern differences. Thus this approach avoids comparison of several 2-D gels.

The drawbacks of 2-DE are: (1) the technique is labour-intensive and time-consuming; and (2) the complete resolution of all proteins does not occur on a single 2-D gel because the protein mixture is too complex to be completely resolved (Graves and Haystead 2002). Large or hydrophobic proteins will not enter the gel during the first dimension, and the pH range of the gel does not permit proteins with isoelectric points (Ip) lower than pH 3 and higher than pH 10 (Gorg et al. 2000) to be resolved. In addition, the gels only show abundant proteins and not low-copy proteins.

An alternative to 2-DE is to digest a protein mixture to peptides by trypsin and then to purify the peptides before analysis by mass spectrometry (MS). The disadvantage is the cost of the instrumentation, the time employed in the analysis and the computing power to deconvolute the data obtained (Graves and Haystead 2002). This approach presents the advantage that it is able to analyse a greater number of proteins than the 2-DE method. In addition, the mass spectrum of the unknown protein can be compared with theoretical mass spectra produced by computer-generated cleavage of proteins in the database (Graves and Haystead 2002).

Another promising alternative to 2-DE is the use of an isotope-coded affinity tag (ICAT) which allows quantitative profiling between different samples without the use of electrophoresis (Gigy et al. 1999). Protein samples are treated with two chemically identical reagents that differ only in the mass as a result of the isotope composition. The ICAT reagent consists of a biotin affinity group, a linker region that can incorporate heavy (deuterium) or light (hydrogen) atoms, and a thiol-reactive end group for linkage to cysteine residues of proteins. This reagent permits the quantification of the expression level of proteins (Graves and Haystead 2002). For example, two sets of populations of cells in a different state can be differentiated because they are labelled with either a light or heavy form of the ICAT reagent, and the difference in peak heights between heavy and light peptide ions directly correlates with the difference in protein abun dance in the cells. In addition, the protein can be identified after hydrolysis by trypsin, purification of labelled peptides by avidin chromatography by virtue of the biotin tag, and analysis by MS.

Another promising technique is microarray systems involving miniature chips and MS, which allow the analysis of a great number of proteins (see Chap. 5).

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