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  2. 2. DEFINITION • Study set of proteins that particular time, under particular circumstances, in biological place such as, cells , tissues or an organism is called proteome. • The word proteome is a blend of protein and genome, and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student. • The proteome is the entire set of proteins , produced or modified by an organism or system.
  3. 3. THE CHALLENGES OF PROTEOMICS • Splice variants create an enormous diversity of proteins • ~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins • Splice variants may have very diverse functions • Proteins expressed in an organism will vary according to age, health, tissue, and environmental condtion • Proteomics requires a broader range of technologies than genomics
  4. 4. REGULATION OF THE HUMAN GENOME Alternative splicing Allows for multiple proteins from one gene.. Also uses different splice sites within introns GTACCGATTGTAGG….AGGGGCTAG Exon 1 Exon 2 Exon 3 Exon 4 Isoform 1 Isoform 2 Isoform 3
  5. 5. DIVERSITY OF FUNCTION IN SPLICE VARIANTS • Example: the calcitonin gene • Gene variant #1 • Protein: calcitonin • Function: increases calcium uptake in bones • Gene variant #2 • Protein: calcitonin gene-related polypeptide • Function: causes blood vessels to dilate
  6. 6. POST-TRANSLATIONAL MODIFICATIONS • Post-translational modifications are defined as any changes to the covalent bonds of a protein after it has been fully translated. • Addition of chemical groups to one or more amino acids on the protein
  7. 7. CHEMICAL MODIFICATIONS • Phosphorylation: activation and inactivation of enzymes • Acetylation: protein stability, used in histones • Methylation: regulation of gene expression • Acylation: membrane tethering, targeting • Glycosylation: cell–cell recognition, signaling • GPI anchor: membrane tethering • Hydroxyproline: protein stability, ligand interactions • Sulfation: protein–protein and ligand interactions • Disulfide-bond formation: protein stability • Deamidation: protein–protein and ligand interactions • Pyroglutamic acid: protein stability • Ubiquitination: destruction signal • Nitration of tyrosine: inflammation
  8. 8. PRACTICAL APPLICATIONS • Comparison of protein expression in diseased and normal tissues • Likely to reveal new drug targets • Today ~500 drug targets • Estimates of possible drug targets: 10,000–20,000 • Protein expression signatures associated with drug toxicity • To make clinical trials more efficient • To make drug treatments more effective
  9. 9. TECHNOLOGIES FOR PROTEOMICS • 2-D gel electrophoresis (2-dimensional) • Separates proteins in a mixture on the basis of their molecular weight and charge • Mass spectrometry • Reveals identity of proteins based on computer software that can uniquely identify individual proteins • Protein chips • A wide variety of identification methods • structure, biochemical activity, and interactions with other proteins • Yeast two-hybrid method • Determines how proteins interact with each other
  10. 10. SEPARATION AND ISOLATION OF PROTEINS • One dimensional gel electrophoresis • Isoelectric Focusing= IEF • Two dimensional gel electrophoresis • High Performance-Pressure Liquid • Affinity Chromatography • Size Exclusion Chromatography • Ion Exchange Chromatography
  11. 11. ONE DIMENSIONAL GEL ELECTROPHORESIS • The work: 1 - Prepare Loading buffer containing sodium dodecyl sulfate and a thiol reducing agent. 2 - Solving the Protein Loading Buffer 3 - binding proteins and sodium dodecyl sulfate complex formation 4 - complexes incorporating sodium dodecyl sulfate - protein into the gel 5 - establish an electric potential between the Jelly Oatmeal 6 - Migration complex 7 - bonds (bond) based on molecular weight proteins
  13. 13. 2-D GEL ELECTROPHORESIS • Polyacrylamide gel • Voltage across both axes • pH gradient along first axis neutralizes charged proteins at different places • pH constant on a second axis where proteins are separated by weight • x–y position of proteins on stained gel uniquely identifies the proteins
  14. 14. CAVEATS ASSOCIATED WITH 2-D GELS • Poor performance of 2-D gels for the following: • Very large proteins • Very small proteins • Membrane-bound proteins
  15. 15. STAINING One of the oldest methods of stained proteins is Coomassie Blue Disadvantage: Extract large amounts protein. Stained with silver nitrate and a more general approach is better because it is more sensitive. Disadvantage: In some cases, this color interact with characteristics of proteins Labeled Before performing the first dimension electrophoresis with radioactive proteins in vitro. Disadvantage: In some cases the staining severely answers
  16. 16. LIMITATIONS OF TWO- DIMENSIONAL ELECTROPHORESIS • It is time-consuming and laborious • The method like PCR for proteins is not accessible and therefore not able to detect proteins with low copy numbers (low expression)
  17. 17. IDENTIFICATION OF PROTEINS • In many cases, knowing the isoelectric point and molecular weight proteins are not adequate for identification, because in some cases, two or more different proteins may have a similar molecular weight and isoelectric. • Edman • Mass Spectrometer
  18. 18. EDMAN • Amine end the desired protein to be sequenced • Alternatively the desired protein from the gel isolated and using enzymatic digestion, peptide fragments were sequenced by Admen. • Disadvantage : large Protein is required and In the end, more than 50% protein, amino end blocked and can not be sequencing.
  19. 19. MASS SPECTROMETER • Proteins Partial sequencing and protein identification • MS technique enables us to get information construct the proteins or peptides, such as MW and amino acid sequences. • This information can be used to search nucleotide databases to identify the protein can be used.
  20. 20. MASS SPECTROMETRY • Measures mass-to-charge ratios of ions • Components of mass spectrometer • Ion source • Mass analyzer • Ion detector • Data acquisition unit
  21. 21. ION SOURCES USED FOR PROTEOMICS• Proteomics requires specialized ion sources • Electrospray Ionization (ESI) • With capillary electrophoresis and liquid chromatography • Matrix-assisted laser desorption/ionization (MALDI) • Extracts ions from sample surface ESI MALDI
  22. 22. MASS ANALYZERS USED FOR PROTEOMICS • Ion trap • Captures ions on the basis of mass-to- charge ratio • Often used with ESI • Time of flight (TOF) • Time for accelerated ion to reach detector indicates mass-to- charge ratio • Frequently used with MALDI Ion Trap Time of Flight Detector
  24. 24. IDENTIFYING PROTEINS WITH MASS SPECTROMETRY • Preparation of protein sample • Extraction from a 2-D gel • Digestion by proteases — e.g., trypsin • Mass spectrometer measures mass-charge ratio of peptide fragments • Identified peptides are compared with database • Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database • Match of experimental readout to database PMF allows researchers to identify the protein
  25. 25. LIMITATIONS OF MASS SPECTROMETRY • Not very good at identifying minute quantities of protein • Trouble dealing with phosphorylated proteins • Doesn’t provide concentrations of proteins • Are only able to identify hundreds of proteins in a single day
  26. 26. PROTEIN CHIPS • Thousands of proteins analyzed simultaneously • Wide variety of assays • Antibody–antigen • Enzyme–substrate • Protein–small molecule • Protein–nucleic acid • Protein–protein • Protein–lipid Yeast proteins detected using antibodies
  27. 27. FABRICATING PROTEIN CHIPS: PHYSICAL ARRAY THAT CAN HOLD PROTEINS, ISOLATE THEM FROM EACH OTHER, AND PREVENT THEM FROM BECOMING DENATURED • Protein substrates: minipads • Polyacrylamide or agarose gels • Glass • Nanowells • Proteins deposited on chip surface by robots Polydimethylsiloxane
  28. 28. READING OUT RESULTS • Fluorescence • Most common method • Fluorescent probe or tag • Can be read out using standard nucleic acid microarray technology • Surface-enhanced laser desorption/ionization (SELDI) • Laser ionizes proteins captured by chip • Mass spectrometer analyzes peptide fragments
  29. 29. DIFFICULTIES IN DESIGNING PROTEIN CHIPS • Unique process is necessary for constructing each probe element • Challenging to produce and purify each protein on chip • Proteins can be hydrophobic or hydrophilic • Difficult to design a chip that can detect both • Protein’s function may be dependent on posttranslational modification or an interaction with another biological molecule
  30. 30. REGULATION OF TRANSCRIPTION TATA boxUE Gene expression requires the following: A DNA-binding domain An activation domain A basic transcription apparatus
  31. 31. YEAST TWO-HYBRID METHOD • Goal: Determine how proteins interact with each other • Method • Use yeast transcription factors • Gene expression requires the following: • A DNA-binding domain • An activation domain • A basic transcription apparatus • Attach protein1 to DNA-binding domain (bait) • Attach protein2 to activation domain (prey) • Reporter gene expressed only if protein1 and protein2 interact with each other
  34. 34. DATABASE BANK • 1. Profound( • 2. MASCOT( • 3.pubmed • 4. Expasy ( • 5.Swiss-Prot ( • 6.IPI (International Protein Index) • 7.SWISS-2DPAGE database
  35. 35. EXPASY SITE
  36. 36. MEDICAL APPLICATIONS OF PROTEOMICS • 1 - Cancer Research • 2 - Infectious Diseases • 3 - Heart disease
  37. 37. BIOMARKER AND CLINICAL APPLICATIONS OF PROTEOMICS • Molecules that show Physiological changes in the cell or tissue or organism and compared to the control sample the simplest definition The biomarker. • Biomarker is biochemical molecules that are able to show a patient and able to measured. • Biomarker can identify disease progression or treatment effect
  38. 38. BIOMARKER FEATURES • Show main feature of the disease, especially in the early stages of the disease. • In comparison with Abnormalities related to the disease are sufficient specificity and sensitivity. • At Laboratory tests can able for identify and trust. • it is noninvasive and relatively easy and inexpensive it is to show.
  39. 39. APPLICATION • Disease Cardiac – Vascular and MI • Schizophrenia • Alzheimer's • Progression of neurodegenerative diseases such as PD and AD • autoimmune Patients • Sickle cell disease(pr in membrane of RBC) • MS disease
  40. 40. FUTURE PROSPECTS • The next decade may see the complete deciphering of the proteome of yeast (done already) • More initiatives, like the Human Liver Proteome Project, are underway • Better understanding of disease: prognosis and diagnosis