Proteome and Proteomics.

The analysis of the total protein content of a cell, or proteome,  has been greatly enhanced by the development of a series of techniques, which together are called "proteomics". A proteomic analysis starts with the selection of a tissue or cell to be studied, and this requires a careful selection of growth conditions together with the standardization and characterization of the sample preparation procedure. Ideally, all the proteins in the samples will be extracted, however in practice, not all protocols will solubilize all the protein fractions of a cell to the same degree, and this is a potential source of error in the technique.  Once the cell preparation protocol has been established, the analytical procedures are adaptations of physicochemical techniques developed in protein chemistry laboratories over last few decades. 

Two dimensional gels separate proteins first according to their  isoelectric point by isoelectric focusing (in the first dimension), and subsequently by molecular mass by SDS polyacrylamide gel electrophoresis (in a second dimension that is perpendicualr to the first).  After the 2D gel is run, it must be treated in a way permits visualization of the protein.  This procedure is called staining, and many staining protocols for polyacrylamide gels have been developed over the last decades.  The staining procedure to be used must be compatible with the subsequent operations, since chemicals or other products added during the staining procedure may act as contaminants during the subsequent mass spectrometry analysis.After the staining procedure is completed, the individual proteins are visible as discrete spots in the polyacrylamide gel. 

After the gel has been stained individual spots can be physically cut out of the gel and extracted from the polyacrylamide matrix.  This is generally associated with the treatment of the material using the proteolytic enzyme trypsin, which generates a mixture of polypeptides which may then be analysed using the mass spectrometry. Since each protein has a particular amino acid sequence, then the mixture of peptides that is derived from this protein will also be unique, and is called a "mass fingerprint".  Once the mixture of peptides has been analyzed by a mass spectrometry, the molecular weights and the amino acid sequences of these peptides may be obtained. 

The peptide mass fingerprints obtained experimentally from the mass spectrometry can be compared with the predicted peptide masses calculated  from in silico translation and tryptic digestion of the protein coding regions in genomic DNA.  This comparison will identify the individual spots in the 2D polyacrylamide gel, and the result of the proteomic analysis is therefore a catalog of all the proteins that were extracted from a given cell or tissue.  This is important information since not all the information encoded in the DNA is used by all of the cells at all times, and proteomic analysis allows the comparison of the protein content of different cells.  Many disease processes are linked to disfunctional regulation of the information processing capability of a cell, and knowing how the pattern of protein expression changes in pathological states may give important clues as to the molecular basis of these diseases.