PROTEOMICS AND BIOINFORMATICS IN LABORATORY MEDICINE : UTILITY FOR DIAGNOSIS AND DRUG DESIGN

Dr. A. R. Aroor

Guest Lecture

Abstract:

Laboratory medicine has witnessed noticeable advances during the last decade through genomics, proteomics and medical bioinformatics. Proteomics is a systematic approach for studying the identity, quantity and function of all proteins expressed in a cell, tissue or organ. The proteome approach includes identification and quantitation of proteins and analysis and interpretation of the proteomics data by informatics. The use of serum or urine samples for proteomics in clinical biochemistry laboratory will explore the identification of biomarkers for disease states. Once the proteome laboratory is established in clinical biochemistry laboratory as a part of molecular diagnostic laboratory, the utility of proteome facility for research is within the reach of the laboratory work for therapeutic proteomics. Studies on alterations in proteome biomarker profile using cell or tissue cultures after exposure to designer drug coupled with animal or human experiments will aim to discover newer drugs. Laboratory and research studies on alcoholism provides a suitable model for proteomics in laboratory medicine comprising (1) proteomic biomarkers for alcohol intake, (2) proteomic biomarkers for alcohol induced organ damage and (3) proteomic biomarkers for drug design.

Proteomics:

Proteomics as an effective tool for molecular diagnosis and drug design compared to conventional laboratory methods. Many of the currently used laboratory methods for the diagnosis and treatment of disease are usually piecemeal analytical methods to assess the function of cell or tissue. However, the sequencing of the complete human genome as well as sequencing of several pathogens has opened the door for new technology in laboratory medicine comprising genomics and proteomics, Genomics and proteomics permit profiling of all the changes that occur in the cells or tissues compared to conventional laboratory methods. Although genomics provide an excellent tool to reveal disease mechanisms and molecular diagnostics, proteomics (the analysis of the protein complement of given tissue or cell) is superior to genomics. While there are about 30000 genes in the human genome, the number of proteins is more than genes because of alternative splicing and exchange of structural cassettes among genes during transcription, leading to two or more effectively different proteins per gene. It is estimated that there are approximately six to seven times as many distinct proteins as genes in humans. Furthermore, proteins are more dynamic in nature than genes because of posttranslational modifications including proteolysis, sulfhydryl oxidation, phosphorylation, glycosylation, S-nitrosation, fatty acylation and oxidation. These posttranslationsal modifications occurring in a normal cell is required for physiologic function of proteins. Alternatively, these posttranslational modifications represent the consequences of environmental modifications of genetic determinants. Thus proteomics is defined as sequence, modifications and functions of all proteins in biological system. Proteomics has the potential to revolutionize the diagnosis of diseases and targeting therapeutic strategies.

Proteome analytical techniques

Two-dimensional electrophoresis (2D PAGE) was the major tool for profiling protein expression during the early years of proteomics. However, several promising new technologies have emerged during the recent years that are more suitable for laboratory medicine. These include protein micro array, non-gel based techniques based on separations of proteins with or without tagging, and direct profiling using mass spectrometry. Two dimensional electrophoresis and mass spectrometry 2D PAGE with immobilized pH gradients allows the production of gels with narrow to wide pH range. Further improvement in 2D PAGE is production of zoom gels. Zoom gel 2D PAGE involves fractionation of individual samples into narrow pH range under low resolution followed by high-resolution separation of individual fraction by 2D PAGE. The successful application of 2D PAGE depends on the availability of powerful bioinformatics approach. At present, several databases are available for mapping 2D PAGE. Affinity capture 2D PAGE is a new technology in which proteins are tagged for the analysis of proteins. A surface protein biotinylation coupled with use of mass spectrometry has led to the identification of many new proteins on the surface of cancer cells.

Non-gel based separation techniques are liquid based separation techniques for proteins with or without tagging have the potential for automation. These liquid separation systems are integrated with mass spectrometry for protein digestion and identification.

Non-separation based techniques include in situ proteomic analysis and protein microarrays. In situ proteomic analysis is direct profiling of proteins using mass spectrometry to obtain the images of normal and disease tissues. In this technique, frozen tissue is sliced and sections are applied on a matrix-assisted laser desorption / ionization (MALDI) plate and analyzed at regular spatial intervals. The mass spectra obtained at different intervals are compared yielding a spatial distribution of individual masses across the tissue section. Mass profiles of tissues sections of normal and diseased tissue will reveal altered protein expression in the diseased state. Protein microarrays using antibody array approaches is a high throughput proteome analysis for clinical proteomics. This technology is further improved by laser capture micro dissection to procure total protein from specific microscopic cellular populations, This technology is also useful for studies involving complex cellular signaling between different cell populations within a tissue. Protein microarray technology has been improved in recent years by development of new strategies for producing biochips that are used for proteome analysis. New classes of capture agents include aptamers, ribozymes, partial molecular imprints and modified binding proteins. Recently, a reverse phase protein array approach has been used for proteome analysis. In this technique, proteins are immobilized followed by detection of proteins by selective antibodies. Protein fractionation coupled with proteome analysis of modified proteins offers functional aspects of proteome and such proteome approach will provide clinical relevant assays of broad sets of protein. This technique requires development of antibodies that recognize specific forms of proteins.

Bioinformatics for data analysis

Bioinformatics for 2D PAGE : Data analysis form 2D-PAGe involves staining, scanning, spot detection and spot quantitation. The data are analyzed using gel matching software application such as MELANE II. Data form 2D PAGE is deposited in 2D databases containing digital images. The databases such as SWISS-2DE and PDQUEST are available in the internet that are indexed at the ExPAsy WORLD-2DPAGE. Software packages such as Make2ddb are available to set up 2D-PAGE databases. Data can be compared using Java programs such as Flicker and CAROL.

Bioinformatics for mass spectrometry The raw data (mass/charge ratios) from mass spectrometry experiments are used to determine accurate molecular masses in peptide mass finger printing or fragment ion searching. Databases and programs are available based on mass of a peptide fragment (Sequest) or amino acid composition (Lutkifisk). Commercial programs such as Mascot (http.www.matrixscience.com) and Sequest (http.www.fields. Scripps.edu/sequest) are useful for analyzing data for unmodified peptides form a database of sequences.

Proteome data analysis is greatly improved by utilizing sources such as metabolic knowledge bases, signal transduction knowledge bases, general aspects from MEDLINE, functional information sources such as LocusLink and SwissProt and protein-protein interaction data from BIND.

Clinical proteomics

Proteomics for screening and diagnosis of diseases Proteomics based screening tests comprise comparison of proteins expressed in tissues or proteins present in biological fluids such as plasma or serum) and urine from patients suffering form particular disease. FDA-NCI (Food and Drug Administration- National Cancer Institute) Clinical Proteomics Program is one of the leading proteomic programs involved in clinical diagnosis and therapy. Studies under this program have shown the significance of plasma proteome profiling in the diagnosis of ovarian cancer and prostate cancer. Studies on myocardial proteins associated with human heart failure and animal model of heart failure have demonstrated changes in set of proteins in altered modifications of proteins such as myosin light chain 2 in the failing heart. Recently, urine proteome profiling after alcohol intake revealed the presence of transferrin as a specific biomarker of alcohol intake.

Proteomics for drug efficacy and toxicity: Although new drugs are being developed, it is very difficult to evaluate their efficacy and toxicity by conventional methods. Sometimes drugs will go as far as human trials before pharmaceutical company realizes that they are either ineffective or have therapeutic toxicity. Proteome analysis approach will offer a systematic approach for studying protein function in diseases and enable faster validation of the new drug.

Drug discovery most of the pharmaceutical companies are focusing on mechanism based drug research with emphasis on drug modulation of protein function. In cancer drug design, proteomic study will enable to explore the protein involved in distinct process of cancer such as initiation, progression and metastasis. Drugs targeting these distinct events will help to develop drugs for prevention, progression or suppression of complications of cancer depending on the stage of the disease present in-patient. Studies on effects on cell signaling have revealed the role of MPAK signaling in alcohol-induced cytotoxicty. The MAPK signaling is a potential target for drug development for alcoholism.

International proteome project and clinical proteomics : Several government and private medical centers have established molecular diagnostic laboratory as a component of laboratory services. Establishing a proteome facility in laboratory medicine will provide facility for better diagnostic services and facility proteome related research. The promising results of proteomics have led to international interest in clinical proteomics. The Human Proteome Organization (HUPO) led by Hanash at the University of Michigan is playing international interaction to map the plasma, brain and liver as well to create antibodies against human proteins. Other tissues and fluids are also included in the proteomics. Plasma proteome project is one of the most advanced project involving 47 laboratories and 13 countries. Plasma proteome is the difficult proteome to map because of its heterogeneous nature but if successful, it is one of the most valuable proteome map available for clinical use. Increased levels of apo-lipoprotein AI were found in the plasma proteome profile after moderate alcohol consumption thus providing an explanation for beneficial cardiovascular effects of moderate alcohol consumption.

In summary, proteomics and bioinformatics in clinical laboratory will emerge as a powerful analytical tool for screening of diseases, diagnosis of diseases, drug monitoring and drug discovery.

References

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