Lecture 23: Clinical and Biomedical Applications of Proteomics; Proteomics Industry
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1 Lecture 23: Clinical and Biomedical Applications of Proteomics; Proteomics Industry Clinical proteomics is the application of proteomic approach to the field of medicine. Proteome of an organism changes as a function of developmental state and diseased condition. Thus, changes observed in the proteome of a diseased individual compared to normal one can be utilized as a biomarker to detect disease, or used as the basis for the development of pharmacological targets for therapeutic intervention. Please recall our discussion during Lecture 17. It is possible to analyze the total protein of normal and diseased person and identify proteins up-regulated/under regulated by peptide mass fingerprinting. This, under or up-regulated protein may act as a biomarker for disease detection. Not only this, the information may also help us in developing therapeutics. For example, if peptide mass fingerprinting results indicate that in a given diseased condition a protease is upregulated, the therapeutic strategy would be to inhibit the protease by developing a specific inhibitor against the protease. IIT Guwahati Page 1 of 6
2 Blood is most easily available sample for proteome analysis. However, unlike other tissues, blood (plasma or serum), in addition to containing proteins specific to blood cell; it also contains proteins released, either through leakage, injury, or other factors, from other tissues in the body. Another difference between blood and other body tissues is the concentration difference of the various protein species present in the blood. For Example: Albumin is present in the milligrams per millilitre range while cytokine, interleukin-6, are present in the picograms per millilitre range. A two dimensional gel electrophoresis data will show prominently albumin in the sample. To have a proper proteome analysis and see expression differences in low abundance protein, albumin has to be removed from the sample by affinity-based methods or other methods. Identification of biomarker by two dimensional electrophoresis and mass spectrometry/ peptide mass fingerprinting has long history. A novel biomarker was identified by comparative proteome analysis of nipple aspiration fluid. Some examples are listed in the table below Table: Biomarkers for various types of cancer identified by proteome analysis. Disease Biomarker IIT Guwahati Page 2 of 6
3 Early prostate and esophageal cancer Annexin-1 Breast cancer Lung cancer Blabber cancer Leukemia HSP27, HSP60, HSP90, PCNA Cytokeratins Keratins, psoriasin Op18, nm23-h1 Efforts towards biomarker discovery have focused on looking for a single over- or underexpressed protein or a disease-induced protein change that is released and detected in the circulation. The poor success in discovering new biomarkers reflects the inability of this approach. There are number of disadvantages of biomarker discovery by proteome analysis. This includes, low sensitivity of the method, and inability to detect alteration in expression of low abundant protein with high confidence. Moreover, there are possibilities of specimen manipulation, problems with the sample collection/sample contamination or pipetting. The Human Proteome Organization (HUPO) established a Specimen Committee in 2002 to study and address many of these issues. Another source of variations that must also be considered in biomarker research is biological variation. Proteome is a dynamic entity and it depends on age, gender, race or even environmental conditions. Thus, for proteomic research and biomarker discovery, biological variations should also be taken into consideration (diseased and healthy individuals used for the comparative analysis should have no or minimum biological variations). A vast range of molecular, cellular and advanced tools are available in modern medical research, which are not only capable of separating proteins on gel but even identifying them. As discussed early during the lecture, proteomics methods are widely applied in Biomarker discovery in human disease, since they allow deep insights into the patho-physiology and prognosis of disorders by identification of altered protein expression. Proteomics have several other biomedical applications. Certain proteomic methods can help in identifying possible function of identified biomarker. High throughput protein-protein interaction studies can be performed to identify interactome of the biomarker to derive possible function for development of therapeutics. Few applications and examples are listed below. IIT Guwahati Page 3 of 6
4 Study on the pathogenesis of human diseases--arsenic carcinogenesis Recent studies have demonstrated that a low level (1.5 μmol/l) of arsenite induces B[a]Ptreated lung cell transformation. Protein Chips were used to identify different protein expression, which could potentially be important for cell transformation induced by this toxic agent. Identification and characterization of these proteins revealed the molecular basis of arsenite-induced cell transformation and helped to elucidate the mechanisms by which arsenic induces carcinogenesis (Journal of Cellular Biochemistry, 2003, 88, 1-8) To identify, characterize and clinically apply biomarkers: Biomarkers of human disease serum in Hepatitis B virus (HBV) infection: HBV is a serious infectious and widespread human pathogen, representing a major health problem worldwide. Chronic HBV infection has a very high chance of evolving into hepatocellular carcinoma. Although considerable progress has been made in the past few years, the pathogenesis of HBV infection is still elusive and a definite diagnosis still relies on biopsy histological test. But recently proteomic technology has been used to examine HBV-infected serum samples in a search for diseaseassociated proteins that can be used as serological biomarkers for diagnosis and/ or target proteins for pathogenic study. The study shows that 2D-PAGE electrophoresis can generate a comprehensive serological profile in which the HBV protein biomarkers change patterns not only in their quantities but also in their qualities. The study found change in expression profile of α1-antitrypsin, DNA topoisomerase II β and few other proteins. (Proteomics. 2003, 3, ) Proteomic studies on chemotherapeutic agents: Certain studies are done in the direction to investigate mechanisms of drug actions. For example: The protein changes in rat livers have been conducted by using various agents including hepatotoxicants, methapyrilene, cyproterone acetate and dexamethasone. Twodimensional polyacrylamide gel electrophoresis and mass spectrometry were used for the identification of compound specific biomarkers. Many of the protein changes are associated with the known pharmacological and toxicological mechanisms of action of the drugs. This approach could open up new avenues for the exploration of molecular mechanisms of toxicity, and is a good illustration of how proteomics can provide valuable information on the biochemical consequences elicited by hepatotoxic drugs. IIT Guwahati Page 4 of 6
5 Proteomics and analysis of protein as a function of growth stage, environmental a condition etc. is also a tool to understand various cellular processes. Proteomics is widely used in Plant Biotechnology as well to proteins of biosynthetic pathways leading to secondary metabolites. Moreover, secondary metabolites are frequently analyzed using mass spectrometry. Many secondary metabilistes are known as drug/drug candidate (Phytochemical Analysis, 2000, 11, ) Recommended article for further reading After the successful sequencing of genome of many organisms, researchers are now headed towards multidimensional and active collection of proteome of organisms. As discussed earlier, in the course, proteome is a dynamic entity and much larger and complex than the geneome (due to alternative splicing of mrna; post translational modification of proteins etc). Proteomics is not only to identify all the proteins of an organism, but also to understand their functions and range of expressions in different cell types. Proteome also include characterization of protein modifications, interactions and structure. Proteome is already yielding drug targets and information on disease states and drug response. Proteomics incorporates protein chemistry and a convergence of analytical approaches like mass spectrometry, spectroscopy, 2-D gel electrophoresis and microarray technologies. Originally, proteomics meant just the isolation and identification of proteins from normal versus perturbed cells. Scope of current proteomics has widened and encompasses aspects of functional genomics (linking protein expression with gene expression), structural proteomics (determining large numbers of protein 3-D structures by spectroscopic or computational techniques), and protein-protein interaction and biochemical pathway studies. Drug developers are urged by the desire to understand disease mechanisms and find protein markers or expression patterns for monitoring drug response and treatment. Further improved target identification and validation is also an area to be explored for better understanding of IIT Guwahati Page 5 of 6
6 cell pathways and communication. Now days the field of pharmaco-genomic and related proteomic data has also opened up for investigation purpose. According to several recent market research reports, the market for proteomics will show double-digit growth over the next four or five years. Among proteomics technologies, the mass spectroscopy market is the largest, with estimated sales of roughly $850 million per year and greater than 10% annual growth. The fastest growing market, at more than 30% per year, is for protein microarrays and chips, now worth about $100 million. Gel electrophoresis is a midsized market at $300 million per year but is expected to grow nearly 15% annually. The protein structure determination methodologies have a market of about $100 million per year and are expected to grow about 10% per year. Developers of proteomics technologies include many small technology and instrumentation firms. Meanwhile, the user base includes both small drug discovery and most large pharmaceutical companies. The big companies are adopting proteomics internally, in core labs or throughout their R&D operations. A survey by bcc Research in June 2009 available online shows potential of proteomics industry. IIT Guwahati Page 6 of 6
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