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Proteomics

  1. 1. PROTEOMICS Sqn Ldr Raka Maitra Guide: Lt Col Harsh Kumar
  2. 2. Introduction windowwabeborogovestaircasedoorjubjub...
  3. 3. Nomenclatures  Genome – sum total of all the genes in an organism.  Genomics – study of sequence of all genes in an organism.  Transcriptome – sum total of all the RNA transcripts an organism can make in its lifetime.  Transcriptomics – study of levels of RNA produced from many genes in a cell at a given time.
  4. 4. Proteomics - Definitions  Proteome – properties and activities of all the proteins that an organism makes in its lifetime.  PROTEin complement to a genOME .  Proteomics - the qualitative and quantitative comparison of proteomes under different conditions to unravel biological processes.
  5. 5. Proteomics - Definition  Proteomics is a scientific discipline concerned with systematic analysis of proteins present in cells at a given time under given conditions.  Proteomics includes the identification, characterization and quantitation of the entire complement of proteins in cells, tissues or whole organisms with a view to understanding their function in relation to the life of the cell.
  6. 6. Protein Synthesis
  7. 7. Why do we need Proteomics?  Level of transcription of a gene ≠ Level of expression of the gene  mRNA - degraded rapidly - translated inefficiently  Post-translational Modifications / Translocations  One gene / transcript > many proteins  One protein > many processes 
  8. 8. Genome Vs Proteome  Information stored in the genome is used differently in different cells  Multigenic diseases  Incorrect modification of a normal protein  Diagnosis of disease  Targets for drugs
  9. 9. Protein Structure Primary structure
  10. 10. Protein Structure Secondary structure
  11. 11. Protein Structure Tertiary structure
  12. 12. Protein Structure Quaternary structure
  13. 13. Technologies in Proteomics  Protein separation  Protein detection  Protein analysis  Identification  Measurement of activity
  14. 14. Protein Separation  2-D polyacrylamide gel electrophoresis (2-D PAGE) :  separation by charge (isoelectric point)  separation by mass  HPLC  Dialysis  Differential Centrifugation  Salting Out
  15. 15. 2-D PAGE
  16. 16. Protein Detection  Staining – non-quantitative  Coomassie blue  Silver  SYPRO Ruby Red  Fluorescence - quantitative  Autoradiography – more sensitive quantitation
  17. 17. Comparison Of Different Protein Detection Methods
  18. 18. Mass Spectrometry  Separates proteins according to their mass-to- charge (m/z) ratio  Ionization of proteins – ions propelled towards the analyzer by electric field - resolves each ion according to its m/z ratio → detector → computer for analysis  Ionization methods :  Matrix-assisted laser desorption/ ionization (MALDI)  Electrospray ionization (ESI)
  19. 19. MALDI-TOF  Ionization by MALDI –  protein suspended in a crystalline matrix  laser energy causes rapid excitation of matrix  passage of matrix and analyte ions into gas phase  ionized protein accelerated by electrostatic field and expelled into a flight tube  Time-of-flight analyzer (TOF) –  when accelerated by application of a constant voltage, the velocity with which an ion reaches the detector is determined by its mass
  20. 20. Electrospray Ionization (ESI)  Production of gaseous ions by application of a potential to a flowing liquid resulting in the formation of a spray of small droplets with solvent-containing analyte  Solvent is removed from the droplet by heat or collision with a gas  Droplet size further decreases to become unstable and explode into even finer droplets  Electrostatic repulsion is sufficiently high to cause desorption of the analyte ions  Passed to the mass spectrometer
  21. 21. Mass Spectrometric Separation Of Different Proteins
  22. 22. Protein Analysis Methods:  Structural Analysis  X-ray Crystallography  Nuclear Magnetic Resonance  Post-translational Analysis – activity based analysis  Newer techniques – analysis of proteins in complex mixtures without separation
  23. 23. X-ray Crystallography  Crystallization:  Supersaturation  Snap freezing  Factors affecting:  pH  Temperature  Precipitant used – salts / polyethylene glycol  Protein concentration
  24. 24. X-ray Crystallography Process:  X-rays directed at crystal of protein / derivative of the protein containing a heavy metal atom  Rays scattered in pattern dependent on electron densities in different portions of the protein  Images translated into electron density maps  Superimposed on one another manually or by specialized computer programs  Construction of a model of the protein
  25. 25. X-ray Crystallography  Disadvantages:  Time consuming  Expensive  Requires very specialized training and equipment  Advantages:  Reveals very precise and critical structural data about amino acid orientation  Used to understand protein interactions
  26. 26. NMR Spectroscopy  Nuclear dipoles in the sample align in a magnetic field  Transmitter pulses radio waves to the sample  Hydrogen nuclei absorb energy and ‘flip’ from one orientation to another  Later flip back and readmit that energy as radio signals  Nuclei in different chemical environments on molecules radiate different energies  Amplified by a receiver and stored on a computer  Software routines interpret the chemical environments
  27. 27. NMR Spectroscopy  Crystallization is not necessary  Facility to reveal details about specific sites of molecules without having to solve their entire structure  Sensitivity to motions on time scale of most chemical events  Adept at revealing how active sites of enzymes work  Transfer Nuclear Overhauser Spectroscopy (TrNOESY) facilitates shape determination of small molecules bound to very large ones, and helps define the binding pocket of the macromolecule.
  28. 28. Post-translational Analysis  Phosphoproteins  Sample digested by proteolytic enzyme alone vs proteolytic enzyme + phosphatase  Phosphoantibodies to precipitate phosphorylated proteins before mass spectrometry  Stains to detect phosphoproteins in polyacrylamide gels  n-linked sugars  Use of glycosylases
  29. 29. Protein Microarrays  Used for:  protein purification  expression profiling  protein interaction profiling  Steps:  Capture  Washing  Uncoupling  Analysis
  30. 30. Newer Techniques  Phage Display:  Creation of peptide or protein libraries on viral surfaces  Peptides or proteins remain associated with their corresponding genes  Cloning of Ligand Targets (COLT)  Alternative to phage display  small peptide sequences bind to larger domain units within proteins  Used to discover new domains and new proteins
  31. 31. Bioinformatics  Building and manipulation of biological databases.  Integration of mathematical, statistical and computer methods to analyze biological, biochemical and biophysical data.  Databases of DNA sequences of genomes. Eg. Genbank, EMBL  Collections of proteomics databases for organisms. Eg. Swissprot, Flybase  Database of computationally derived protein structures.
  32. 32. Proteomics in Life Sciences Proteomics has many diverse practical applications in the fields of:  Medicine  Biotechnology  Food sciences  Agriculture  Animal genetics and horticulture  Environmental surveillance  Pollution
  33. 33. Applications in Medicine • Protein changes during normal processes like differentiation, development and ageing • Abnormal protein expression in disease development (especially suited for studies of diseases of multigenic origin) • Diagnosis • Prognosis
  34. 34. Applications-contd. • Identification of novel drug targets • Selection of candidate drugs • Surrogate markers • Targets for gene therapy • Toxicology • Mechanism of drug action
  35. 35. Applications  Understanding gene function  Understanding the molecular regulation of the cell  Identification of multiprotein complexes  Studying cellular dynamics and organization  Studying macromolecular interactions
  36. 36. Type II Diabetes at the Molecular Level  Aim: Human skeletal muscle is being analysed to find proteins whose expression correlates with the development of T2D.  Project design: Comparison of healthy and diabetic persons of normal and obese build.  Sample treatment: Punch biopsies are collected and snap frozen or rapidly transferred to CPA for labelling with [35 S]-methionine.
  37. 37. T2D  Results so far: Several markers for T2D development have been identified and patented. Post translational Modifications play a decisive role in the development of the disease.  Significance: Modulation of the expression of these proteins, this might offer a new treatment for diabetes.
  38. 38. Mechanism Behind Rheumatoid Arthritis  Aim: Human synovial fluid (including the cells therein), the surrounding tissues and serum are being analysed with the aim to identify pathophysiological changes that can be used diagnostically or therapeutically.  Project design: Comparison of synovial fluids, biopsies and sera from persons at different stages during the development of arthritis.  Sample treatment: Biopsies and cells are collected from synovial fluid, labelled with [35 S]-methionine and tested with sera from arthritic patients.
  39. 39. RA  Results so far: This approach has allowed us to identify early antigens and antibodies in the synovial fluids.  Significance: Early results suggest that this may allow us to determine the effectiveness of treatment.
  40. 40. Changes That Occur During Ageing  Aim: Human skin biopsies are being studied to reveal changes in the physical structure of skin in order to chart the changes that occur with age so we will be able to develop treatments which will retard the process or protect it from environmental stress.  Project design: Comparison of protein expression patterns in human skin biopsies from persons at different ages, different sites on the body and of different gender.  Sample treatment: Skin biopsies are collected, labelled with [35 S]-methionine.
  41. 41. Ageing  Results so far: An extensive database is being built up and some markers have already been identified.  Significance: Ageing is something that no one can avoid. Therefore, these results have applications not only in the cosmetic industry, but also in many other fields, because our skin is very active. Eg. in the excretion of waste products; the regulation of temperature; the protection from harmful radiation; and the uptake of certain types of medication.
  42. 42. Colon cancer  Aim: Proteomics is also being used here to carry out a search for molecular markers, which could predict prognosis from pre-malignant to malignant disease and predict efficacy of cytotoxic therapy in a reliable way.  Project design: Human biopsies of colorectal tissue are collected at different stages of cancer development and compared to identify progression markers.  Sample treatment: Colorectal tissue biopsies are labelled with [35 S]-methionine.
  43. 43. Colon cancer  Results so far: We have developed procedures by which bacterial infections can be avoided so that this does not influence the analysis of the polyps. A number of surprisingly large changes have been selected and the proteins identified.  Significance: There are no reliable methods that can be used in predicting the response of patients to radiation or chemotherapy. Considering the increasing number of persons affected and the treatment options available, a convenient non-invasive diagnostic kit would be of great value.
  44. 44. Pathogenesis of Cholesteatoma  Aim: Testing the two theories: whether the hyperkeratinization of this destructive middle-ear disease is due to changes in the lipid metabolism or whether it is due to bacterial infection or both. If it is the former, the goal is to identify which metabolic pathways have failed, and if the latter, the goal is to determine which microorganisms are present, and whether they are the direct or indirect causative agents or only opportunistic infections.  Project design: Biopsies are collected and divided into the pathologically distinct parts of the epithelium and compared against normal skin from the ear canal.
  45. 45. Cholesteatoma  Sample treatment: The various parts are labelled with [35 S]-methionine.  Results so far: A number of striking differences has been identified which suggest that bacteria are not directly involved in the aetiology of the disease.  Significance: Direct treatment would spare the patients for surgical intervention.
  46. 46. Free Radicals in Ischaemia and Thrombosis  Aim: Characterization of the damage caused by free radicals in human blood following for example acute disorders like ischaemia, or chronic disorders like vasoconstriction, and their role in the development of thrombi. The ultimate goal is to find points at which the process could be regulated or the detrimental effects alleviated.  Project design: The effect of anoxia and reperfusion will be investigated on isolated blood vessels and cells to follow the development of oxidative damage. This project will then compare the effects seen in man with those seen in an animal model.
  47. 47. Free Radicals  Sample treatment: A combination of fluorescent labelling and [35 S]-methionine labelling will be used depending upon the type of sample. All samples will be analysed by 2DGE.  Results so far: Reaction pattern of the granulocyte has been intensively studied and several reaction pathways have been characterised. Resistance arteries have been studied, and proteins whose expression correlates to hypertension have been identified.  Significance: Cardiovascular diseases are one of the
  48. 48. Drug Proteomics  Unrecognized connections between proteins and protein complexes, drugs, and biological processes are identified with proprietary proteomics technologies.  Ability to select druggable targets, choose lead molecules with key features, and reject targets with safety concerns.  Rational framework to elucidate mechanism of action for bioactive molecules.  Bioinformatics - translating experimental data into mechanistic models of cellular function and dysfunction and ways to interfere using compounds and drugs.
  49. 49. Environmental Proteomics  Studies of the health effects of environmental agents.  Many environmental chemicals interact directly with cellular protein to modify their functions and interactions.  Environmental agents also may affect gene expression and the levels of protein products of those genes.  Proteomics technologies used to investigate the interplay of environmental agents and the proteome.
  50. 50. Microbial Proteomics  Bacterial genomes encode all possible virulence determinants, vaccine candidates, and potential drug targets.  A completed genomic sequence allows high throughput analysis of the proteome.  Mycoplasma pneumoniae - second smallest genome of any self- replicating life form and encodes 679 putative proteins.  Genome- predicted proteins correlated with those actually present, detecting any biological event that generates a protein of different molecular composition than that predicted.
  51. 51. Recent Advances  Reverse Proteomics  Starting point is the DNA sequence of the genome  Transcriptome and proteome are predicted in silico  This information is used to generate reagents for their analysis.  Shotgun Proteomics  Complete bypassing of  2D-gel electrophoresis  Enabled by Multidimensional Protein Identification Technology (MudPIT).
  52. 52. Conclusion

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