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7.4 Proteomics

Traditionally, the study of the biochemistry or structure of a protein necessitated its purification to a high degree. The development of protocols for cloning, manipulation and expression of genes greatly facilitated this kind of study, as will be clear to you from the previous section. In recent years, a number of high-throughput techniques have, to an extent, obviated traditional approaches and permit simultaneous analysis of all the expressed proteins in a cell or organism, known as the proteome. The term proteomics has been coined to describe such studies.

Proteomics is founded on the technological achievements of genomics, the large-scale analysis of every gene in an organism, and encompasses all aspects of protein structure and function, including post-translational modifications and interactions, and comparisons not only between tissues in an organism but within a single cell under different conditions or at different stages in the life of the cell or organism.

Unlike the genome of an organism, which remains largely unchanged in the lifetime of the cell or organism, the proteome is highly dynamic. Thus, the proteome varies as expression of genes is switched on or off in response to stimuli and the level of expression of different genes is modulated, whilst post-translational modifications and protein interactions add further layers of complexity. As well as identifying and determining the physiological role of proteins, the proteomic approach can highlight changes that occur in pathological situations and can facilitate identification of targets for therapeutic intervention in disease.

Table 7 details some of the technologies and methodologies on which proteomics is based, some of which have been described in previous sections.

Table 7 Methodologies used in proteomics.

Methodology Description Application
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) Proteins are separated in one dimension on the basis of their charge in a pH gradient (isoelectric focusing) and in the second dimension on the basis of their mass (Figure 53). Separation of many thousands of proteins from each other; allows purification of individual proteins and their modified forms.
Mass spectrometry (e.g. MALDI-TOF) A mass spectrometer separates molecular species according to their mass / charge ratio. Can be used on small quantities. For example, proteins eluted from 2-D gels and subjected to proteolysis can be analysed in this way. See also Section 7.3. Accurate mass and sequence information for peptides to identify all proteins present in any sample. Allows comparisons between, for example, diseased and normal tissues. Analysis of post-translationally modified forms of proteins; e.g. comparisons between proteins after treatment with glycosylases or phosphatases, or after use of antibodies specific for modified forms.
Protein microarrays Up to 10 000 purified proteins can be immobilised on a glass slide under conditions that preserve the proteins' native conformations, allowing them to be ‘probed’ in a number of different ways. Binding is read and interpreted automatically. Interacting proteins can be eluted for analysis, e.g. by mass spectrometry. To rapidly screen for protein–protein interactions using a fluorescently labelled protein. To rapidly screen for enzyme–substrate interactions, e.g. identifying target proteins for a particular kinase using radio-labelled phosphate. To examine binding of other ligands including nucleic acids, carbohydrates, receptors, antibodies or even components of whole cell extracts.
Interaction studies e.g. two-hybrid system and phage display See Section 7.3. These library-based methods allow large numbers of proteins to be screened for interactions.
Bioinformatics A combination of mathematical, computer and statistical methods to analyse biological data. Proteomics databases exist for an increasing number of model organisms. Such databases can assist in the identification of proteins and the prediction of their function. They are also used in drug design.

By way of example, Figure 53 illustrates one type of experiment that is routinely performed to assess the biological effects of a treatment at the proteomic level, namely two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with mass spectrometry.

Figure 53 Comparative 2-D PAGE of extracts prepared from (a) control (untreated) and (b) treated cells. The first separation was performed by isoelectric focusing, which separates proteins according to their charge in a pH gradient. The lane containing the separated proteins was then cut from the gel and overlaid on another gel and proteins were further separated by SDS–PAGE (i.e. according to mass). A comparison of the two gels reveals some distinct differences, such as those highlighted in the enlarged regions. The proteins in these spots were eluted and analysed by MALDI-TOF. The experimental amino acid sequence data were then used to search a database for matching protein sequences. In this way, the identity of the proteins in the three positions indicated was determined. Two of the spots in gel (b) correspond to protein X. Thus there is evidently a change in the M r of this protein in treated cells. This shift may result from post-translational modification. There is an increase in the level of protein Y in the treated cells compared to the untreated cells.

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