Out of the two methods of genome-wide analysis, proteomics, the examination of the complete set of proteins synthesized by a cell under a given set of physiological or developmental conditions, should be most informative when making functional assignments. This is mainly due to the fact that the proteome is context-dependent and unlike mRNAs, proteins are functional entities within the cell. The proteome is the proteins expressed by a genome.
Proteomics involve the analysis of a large number of proteins with a combination of two-dimensional gel electrophoresis and mass spectrometry. Today, it includes both the identification of a large number of proteins expressed by a genome as well as the characterization of their functional and structural relationships. It was thought that a single gene would result in the formation of a single protein or polypeptide. But there are different stages in the process of transcription and translation, where the same process can take place but produces a different protein. These processes can take place just after the process of transcription-the post-transcriptional process that can result in different types of mRNAs with the control of splicing mechanisms. The second point where the alteration can occur is after the process of translation or protein synthesis. This is the post-translational modification which can also produce structurally and functionally different proteins from the same gene.
Proteomics also deals with the use of qualitative and quantitative protein-level measurements to characterize biological processes (e.g., diseases). By comparing the protein profiles from normal cells and diseased or metabolically aberrant cells, the reason for the diseased condition can be ascertained. Therefore, diseases can be treated at the protein level or even at the gene level because the proteins are the drug targets.
The Key Technologies in Proteomics
1) Reproducible Two-dimensional Gel Technology
2) Staining and Scanning Technology
3) Mass Spectrometry for Identification
4) Databases (protein and genome)
5) Database Searching Algorithms
The crude protein extracted from the tissue sample or cells or from individuals is separated in gel by two-dimensional electrophoresis comprising of IEF and SDS-PAGE. The gel is suitably stained and compared with that of the standard two-dimensional gel profile of protein from the same organisms grown under normal conditions (or healthy individuals). If the formation of any new protein is observed that spot can be excised from the gel along with the gel matrix and washed with sterile distilled water. This protein spot along with the gel can be digested with trypsin and then introduced into the mass spectrometer and (the MS fingerprint can be taken). The MS fingerprint can be used for the identification of the protein by database search or can proceed further to carry out amino acid sequencing by mass spectrometer. Thus, the protein purification and identification become very easy and simple. But the key challenge in proteome research is the automation and integration of these technologies.
Tags: Bio Technology, Bio Genetics , Proteomics
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