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Exploring Proteins and Proteomes

Exploring Proteins and Proteomes. Genome. A collective name for the genes existed in an organism C. elegance (roundworm) : 97 million bases, 19,000 genes Drosophila melanogaster (fruit fly) : 180 million bases, 14,000 genes Human : 3 billion bases, 25,000 genes

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Exploring Proteins and Proteomes

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  1. Exploring Proteins and Proteomes

  2. Genome • A collective name for the genes existed in an organism • C. elegance (roundworm) : 97 million bases, 19,000 genes • Drosophila melanogaster (fruit fly) : 180 million bases, 14,000 genes • Human : 3 billion bases, 25,000 genes • Static and absolute information Proteome • A collective name for the proteins expressed by the genome • Dynamic and functional information • It varies with cell type, developmental stage, and environmental condition such as the presence of hormones. • Regulation of mRNA synthesis, alternative splicing, mRNA stability, rate of protein synthesis, post-translational modification, protein stability control, protein degradation

  3. Protein Purification • The purification of proteins is an essential first step in understanding their function. • Purification should yield a sample of protein containing only one type of molecule of interest. • Proteins can be separated from one another on the basis of solubility, size, charge, and binding ability. • Assay : a test for some unique identifying property of the protein • Specific Activity : the ratio of enzyme activity to the amount of protein in the enzyme assay NADH can absorb light at 340 nm. Nicotinamide adenine dinucleotide

  4. Homogenation and Fractionation by Centrifugation

  5. Salting Out & Dialysis • Salting Out : protein solubility decrease by very high concentration of salt • Salting In : protein solubility increase by low concentration of salt • Dialysis : separation of small molecules from proteins through membrane with pores such as cellulose membrane (cf. semi-permeable) Ammonium sulfate for protein precipitation

  6. Gel-Filtration Chromatography (Molecular Exclusion, Size Exclusion, Molecular Sieve) • Thyroglobulin (669 kd) • Catalase (232 kd) • BSA (67 kd) • Ovalbumin (43 kd) • Ribonuclease (13.4 kd)

  7. Cation Exchanger Negatively Charged Column Positively Charged Proteins Ion-Exchange Chromatography Anion Exchanger Positively Charged Column Negatively Charged Proteins Depend on local charge on proteins

  8. Affinity Chromatography (concanavalin A) Highly specific - His tag LC Liquid Chromatography FPLC Fast Pressure Liquid Chromatography HPLC High Pressure Liquid Chromatography

  9. v = Ez / f v : velocity of migration E : electric field strength z : net charge on the protein f : frictional coefficient f = 6r  : viscosity of the medium r : radius of the protein Gel Electrophoresis Polyacrylamide gel electrophoresis

  10. Ammonium Persulfate Polymer Formation of Acrylamide using Bis-Acrylamide for PAGE (Poly-Acrylamide Gel Electrophoresis) Sieving action!

  11. SDS-PAGE : Denaturing Gel (Determination of the Molecular Weight of Protein) Coomassie Blue Staining; > 0.1 mg) (cf. Silver Staining: > 0.02 mg) Except carbohydrate-rich proteins, membrane proteins Mobility ; Log of MW Resolution: 2% MW difference Under BME, DTT One SDS anion for every two a.a.

  12. pI : Isoelectric Point (pH with net charge zero) Isoelectric Focusing & Two Dimensional Electrophoresis + - + -

  13. As purification continues, relative presence of contaminants should be decreased and the proportional amount of the protein of interest should be increased. Evaluation of Protein Purification

  14. Centrifugation & Sedimentation Coefficient • s = m(1 - v) / f • s : sedimentation coefficient • m : mass of the particle • v : partial specific volume; the reciprocal of the particle density • : density of the medium (1 - v) : buoyant force exerted by liquid medium f : frictional coefficient; a measure of the particle shape • A more massive particle sediments more rapidly. • A more compact shaped particle sediments faster. • (i.e. elongated particles sediments more slowly than do spherical ones of the same mass. Frictional coefficient f) • A denser particle sediments more rapidly. Buoyant force is smaller for the denser particle • v < 1 : sink, v > 1 : float, v = 1 : no movement

  15. a. Seidmentation process in the cell Principle of Analytical Ultracentrifugation From Archimedes’ principle Buoyancy = weight of displaced fluid Or fluid density x submerged vol x g

  16. b.solute distribution in the cell

  17. S Value for Various Proteins

  18. Density and Sedimentation Coefficient for Various Cellular Components

  19. Gradient (Zonal or Band) Centrifugation: Separation of Non-Denatured Proteins with different sedimentaion coefficients (Size, Density and Shape) Sedimentation velocity • Sedimentation equilibrium: centrifuged at low speed so that sedimentation is • counter balanced by diffusion • Very accurate in mass determination without denaturing. • useful for large multimeric proteins.

  20. (Asp, Gly2, Ala, Phe, Arg) Determination of Amino Acid Composition of the Peptide • Peptide hydrolyzation by heating it in 6N HCl at 100oC for 24 hrs • Ala-Gly-Asp-Phe-Arg-Gly 2. Separation of amino acid hydrolysates by ion-exchange chromatography (e.g. sulfonated polystyrene resin; Dowex-50)

  21. 3A. Quantitation of Each Fraction by Ninhydrin; Yield Visible Color (usually blue except Pro for yellow); Detection Sensitivity = Microgram (10 nmol) of an Amino Acid 3B. Quantitation of Each Fraction by Fluorescamine; Yield Fluorescence; Detection Sensitivity = Nanogram (10 pmol) of an Amino Acid

  22. (FDNB) Yield Fluorescent Sulfonamide Identification of N-Terminal Amino Acid FDNB, Dabsyl Chloride, or Dansyl Chloride Can Specifically React with the N-terminal Amino Group, and Yield DNB-Amino Acid, Dabsyl Amino Acid, or Dansyl Amino Acid, and These Can Be Identified by Their Chromatographical Properties.

  23. Determination of Amino Terminal Residue of a Peptide using Dabsyl Chloride

  24. Edman Degradation Sequentially Removes One Residue at a Time from the Amino End of a Peptide up to 50 times Each round can be complete within 1 hr and the Edman degradation can be repeated up to 50 cycles in Practice.

  25. Separation of PTH-Amino Acids Current Sensitivity of PTH-AA Detection Using Gas-Phase Sequenator: Picomole Phenyl Isothiocyanate (PITC) Can Specifically React with the N-terminal Amino Group, and Yield Phenyl Thiocarbamoyl (PTH) Amino Acid, and This Can Be Identified by Its Chromatographical Property. Mild acidic condition

  26. For sequencing of an entire Protein…?? Divide and Conquer !!!

  27. Deduction of Full Amino Acid Sequence of a Protein by Overlapping the Sequences Obtained from individual Peptides

  28. The sequence of a protein of interest can be compared with all other known sequences to ascertain similarities. (Family, function prediction possible) • Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathway. • Amino acid sequences can be searched for the presence of internal repeats. • Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing. (N-terminal 20 hydrophobic residues, signal sequence, nuclear localization signal) 4 Repeating Motifs in Calmodulin : Each Unit Binds a Calcium Ion The Amino Acid Sequence Provides Insights into the Protein’s Function, Structure, and History

  29. Antibody • Antibody (immunoglobulin) is a protein synthesized by an animal in response to the presence of a foreign substance (antigen). • Antibodies have specific and high affinity against antigens. • Proteins, polysaccharides and nucleic acids can be effective antigens. • Epitope : a specific group or cluster (portion) of antigen to stimulate the synthesis of an antibody and recognized by a specific antibody (antigenic determinant) • Hapten : a small molecule containing epitope attached to a carrier

  30. Antibody (continued) • Each antibody producing cell synthesizes only one type of antibody recognizing a single kind of epitope. • The proliferation of a given antibody producing cell is stimulated by the binding of its designated antigen to the cell surface receptor of the antibody producing cell . • Periodic injections of an antigen into the host animal can raise the antibodies specifically recognizing the injected foreign substance. • Blood withdrawn from the immunized host animal  centrifugation  separation of blood cells (pellet) and serum (supernatant)  anti-serum • Anti-serum contains multiple kinds of antibodies each recognizing a different surface feature of the same antigen. • This heterogenic antibodies are called as polyclonal antibodies. • This heterogeneity can complicate the use of these antibodies.

  31. Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies. • Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin) Immuno- Staining of Drosophila Embryo using Monoclonal Antibody against Engrailed Monoclonal Antibody Plasma cell by antigen-antibody interaction

  32. Monoclonal antibody drugs?

  33. Marketable? VEGF-A TNF-A CD20 TNFR VEGF Figures from: http://www.patentdocs.org/2009/06/future-drug-sales-predictions-highlight-importance-of-followon-biologics-legislation.html

  34. Antibody naming

  35. Examples of anticancer monoclonal antibody

  36. Herceptin Binds to the C-terminus of Domain IV HER2 Herceptin Fab II I N III IV C

  37. Surface representations of EGFR and HER2 in Antibody-Bound Conformations

  38. ELISA (Enzyme-Linked Immuno-Sorbent Assay) Antibody detection, anti-HIV antibody Antigen detection

  39. Western Blotting Radioactive secondary antibody For protein expression and purification

  40. Immuno-Electron Microscopy Immuno-Fluorescence Microscopy Detection of a channel protein from the synaptic vesicles using antibodies tagged with electron-dense markers such as gold or ferritin (Resolution better than 10 nm) Actin Filament Staining using a-actin antibody Fluorescence-labeled antibodies (resolution 200nm) ex) Glucocorticoid receptor

  41. Synthetic Antigens for antibody formation • Receptor or Interacting Protein Isolation • Clinical Drugs (ex, vasopressin) • 3D Structure Study Synthetic Peptides

  42. MALDI-TOF Mass Spectrometry MALDI : Matrix-Assisted Laser Desorption-Ionization TOF : Time of Flight F=ma Mass Spectrometry Are Often Combined with 2D Electrophoresis for Proteome Analysis

  43. Electrospray Ionization (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Primary Ionization Techniques for Molecules

  44. + + Laser + m m m a + a a m m m m + m a a a m matrix + analyte m Sample support Matrix Assisted Laser Desorption Ionization (MALDI)

  45. AH+ Matrix Assisted Laser Desorption Ionization (MALDI) Laser • 1. Sample (A) is mixed with excess matrix (M) and dried • on a MALDI plate. • 2. Laser flash ionizes matrix molecules. • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. Sample plate hn Variable Ground Grid Grid +20 kV

  46. 40000 30000 20000 10000 0 MALDI/TOF Mass Spectrum (M+H)+ Relative Abundance (M+2H)2+ (M+3H)3+ 50000 100000 150000 200000 m/z

  47. Matrix

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