PROTEOMICS is the study of proteins at the molecular level.

Study of proteins

1. OUT LINE Introduction Protein Synthesis Structure of Proteins Central Dogma Types of Proteomics Importance of Proteomics Principles and Tools of Proteomics Applications of Proteomics

2. PROTEOMICS

3. Proteomics is defined as a comprehensive study of the entire protein composition that is characteristic for a particular Physiological or Pathological state of the cell or whole organism. Human Body contains 2million different Proteins each having different functions.

4. Structure of Proteins Primary Structure---Linear Sequence of Amino Acids joined together by Peptide Bonds in a Protein.

5. Secondary Structure Locally folded regions of the Primary polypeptide chain into regular structures like Alpha Helix, Beta sheets turns and loops.

6. Tertiary Structure Three dimensional compactly folded structure of Proteins and it represents overall organization of secondary structure in 3D space

7. Quaternary Structure Interaction between individual Protein subunits, polypeptide chains assemble to form multi subunit structure.

8. Central Dogma

9. Types of Proteomics

10. Studies the changes in Protein Expression both qualitatively and quantitatively. It detects variations in Protein Expression in different cells such as tumour tissue compared to normal tissue. The Protein expression “ Signature” is conceptually similar to that obtained by Genomic Microarray and allows investigators to identify Biomarkers and Therapeutic Targets. Technique used are 2D Gel Electrophoresis and Mass Spectometry to detect and quantify Proteins.

11. Concerned with determining the 3D structure of Proteins . Techniques used are X-ray crystallography and Nuclear Magnetic Resonance Spectroscopy (NMR) to determine the precise arrangement of atoms within a Protein.

12. Focused on understanding the functions of Proteins and their interactions with other molecules in the cell. It involves identifying the Protein Complexes and interactions that are involved in specific Biological processes and also identifying post Translational Modifications. Techniques used are Advanced MS technologies, Protein Microarray and bioinformatics tools to analyze and interpret large-scale proteomic data.

13. Importance of Proteomics

14. Principles and Tools for Proteomics

15. Steps involved in Proteomic Analysis.

16. Samples Fresh or Snap Frozen cells of Biological fluids such as Serum, Urine or tissue samples. Paraffin embedded and formalin fixed tissue are also used. Tissues obtained from Laser Capture Microdissection of Human colon cancer and HCC have been successfully used.

17. Extraction

18. Separation

19. Mass Spectrometry

20. Ionization source Two common Ionization Technologies --MALDI ( Matrix Associated Laser Desorption Ionization) -- Electron Spray Ionization

21. MALDI ( Matrix Associated Laser Desorption Ionization) Samples are incorporated into a chemical matrix, laser activation of the targets leads to Release of Ions More recently MALDI is replaced by SELDI( Surface Enhanced Laser Desorption Ionization) with different surfaces with strong affinity for Phosphorylated Proteins is introduced.

22. Electron Spray Ionization Peptide ions are generated from aqueous solution Solution containing the sample is passed through a needle with high voltage, spray droplets are passed through cathode to sampling cone.

23. Mass Analysers Work on the Principle of measuring Mass to Charge Ratio( m/z) Types

24. Detector The Mass Spectrum Produced by the Mass Analyser are compared(by using computer algorithms) to a data base of theoretical mass spectra of all known proteins for protein identification.(BIOINFORMATICS) This allows identification of the unknown protein.

25. MS based Functional Proteomics Protein Expression Profiling ---- For identifying Biomarkers that aid in diagnosis. ---- Uses SELDI TOF MS technique. ---- Advantage is it requires only a small quantity of Biologic fluid/tissue.

26. Differential Protein Profiling ( 3 Types) ---2D GE along with MALDI-TOF ---Stable isotope labelling in cell cultures --- Isotope coded affinity tag

27. Imaging Mass Spectrometry Emerging technique which identifies Biomarkers( by MS) directly on tissue biopsies. Frozen tissue is sliced and sections are applied on a MALDI Plate and analysed at regular interval. Analysis using this approach have revealed up to 1600 protein from histologically selected regions of single frozen sections.

28. High throughput method used to track interactions and activities of Proteins and to determine their functions on a large scale. It involves immobilizing proteins on a solid surface, then probing with various analytes to study protein-protein interactions, enzyme substrate relationships. Useful in Biomarker discovery, diagnostics and drug development. Advantage is small sample requirement and ability to simultaneously analyse many proteins in parallel.

29. Clinical Proteomics Cancer Diagnostics. Recent Proteomics studies has showed promising results in Ovarian Cancer using SELDI-TOF MS by identifying 3 biomarkers. HCC- --3 Proteins were found to be overexpressed hep 70 , Peroxidoxin and Manganese Superoxide dismutase (Mn-SOD)—Potential markers for HCC. LUNG Ca--- Napsin A Protein was only expressed in Patients with primary lung Adeno ca, used as a potential biomarker to differentiate the primary from metastatic Adeno Ca.

30. 2D –GE method was used to identify new marker of ALL—heat-Shock Protein 27 (HSP 27). Using SELDI TOF MS serum proteomic profiles from patients of FAP/Celecoxib clinical trials, Celecoxib was shown to be efficacious in cancer prevention . Extension of Proteomic approach for the analysis of drug resistance is emerging as an exciting field.

31. Cardiovascular disorder Cardiomyopathies have received special attention from Proteomics researchers. Decreased expression of 88 myocardial proteins in humans with Dilated Cardiomyopathy, suggested that these low abundant proteins are considered as useful diagnostic and prognostic markers for DCM

32. Cardiac allograft rejections , posttransplant endomyocardial biopsies have shown that 100 proteins were over expressed after cardiac transplantation, of which 13 proteins had cardiac tissue specificity, of these 2 proteins ( alpha-beta crystallin and tropomyosin) could be measured in patient serum,

33. Infectious Diseases Tuberculosis - to improve immunodiagnostics and vaccines for Mycobacterium TB, studies have shown antigens rRV3369 and rRV3874 had high specificity. These can be used in serodiagnosis of TB. To study Protein expression in virulent and avirulent strains, drug resistance of microorganisms and host immune response to infection. Proteomic analysis of sera from SARS patients identified consistent increase in truncated alpha 1 antitrypsin , that could be used as a specific diagnostic marker and also for drug targets.

34. Summary In combination with Genomics Proteomics can provide a Holistic understanding of the biology underlying disease process Information at the level of Proteome is critical for understanding the function of specific cell types and their role in health and disease. Proteomic technologies can also be used to identify markers for cancer diagnosis to monitor disease progression and to identify therapeutic targets.

35. As new protein biomarkers are discovered through Proteomic approaches the necessity to validate and ultimately use them in a clinical setting increases. The National Cancer Institute has taken the lead role in this regard by creating the Early Detection Research Network. This network brings together national and international experts from academic and industry to promote biomarker discovery and validation and help translation into clinical practice.

36. References Philip T Cagle. Timothy Craig Allen. BASIC CONCEPTS OF MOLECULAR PATHOLOGY . Richard A McPherson, Mathew R Pinus HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT BY LABORATORY METHODS 24 th edition. Megan S Lim and Kojo SJ Elenitoba -Johnson PROTEOMICS IN PATHOLOGY RESEARCH. Ramdas Nayak, Rakshata Nayak REVIEW OF POSTGRADUATE PATHOLOGY GENERAL PATHOLOGY. Various Internet sources

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