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Chapter 9-3 From Genomes to Proteomes

Lehninger Principles of Biochemistry, Fourth Edition, 2005. Chapter 9-3 From Genomes to Proteomes. 中央研究院 生物化學研究所 曾湘文 博士 April 03, 2007. Revolutionary technologies in Biology. 1. Restriction enzymes 2. Cloning 3. DNA sequencing 4. PCR (Polymerase chain reaction) 5. SNPs and RFLPs

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Chapter 9-3 From Genomes to Proteomes

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  1. Lehninger Principles of Biochemistry, Fourth Edition, 2005 Chapter 9-3From Genomes to Proteomes 中央研究院 生物化學研究所 曾湘文 博士 April 03, 2007

  2. Revolutionary technologies in Biology • 1. Restriction enzymes • 2. Cloning • 3. DNA sequencing • 4. PCR (Polymerase chain reaction) • 5. SNPs and RFLPs • Single-Nucleotide Polymorphisms • DNA fingerprinting-Restriction fragment length polymorphisms • 6. Oligonucleotide mutagenesis • 7. CHIPs (DNA arrays) • 8. Systems biology

  3. Human Genome Project (HGP) Clones isolated from a genomic library were ordered into a detailed physical map, then individual clones were sequenced by shotgun sequencing protocols. The strategy used by the commercial sequencing effort eliminated the step of creating the physical map and sequenced the entire genome by shotgun cloning.

  4. Genomic sequencing timeline • Discussions in the mid-1980s led to initiation of the project in 1989. • Preparatory work, including extensive mapping to provide genome landmarks, occupied much of the 1990s. • Separate projects were launched to sequence the genomes of other organisms important to research. • The first sequencing efforts to be completed included many bacterial species (such as Haemophilus influenzae), yeast (S. cerevisiae), a nematode worm (C. elegans), the fruit fly (D. melanogaster), and a plant (A. thaliana). • Completed sequences for mammalian genomes, including the human genome, began to emerge in 2000. Each genome project has a website that serves as a central repository for the latest data.

  5. Snapshot of the human genome • Only 1.1% to 1.4% of human genome DNA actually encodes proteins. • More than 50% of genome consists of short, repeated sequences, • The vast majority of which—about 45% of genome in all—come from transposons, short movable DNA sequences that are molecular parasites.

  6. DNA microarray

  7. Outline • The characters of proteins • Why to study proteome • Proteomics • Introduction to proteomics • The major techniques in current proteomics • Post-translational Modification • Protein-protein interaction • proteomics database • Major Directions in Coming Proteomics

  8. Synthesis of DNA and protein DNA Proteins Sugar Chain

  9. Mechanisms by which a single gene can give rise to multiple gene products

  10. Three developments formed the foundation of the new biology • The growth of gene, expressed sequence tag (EST), and protein-sequence databases during the 1990s. • The introduction of user-friendly, browser-based bioinformatics tools. • The development of oligonucleotide microarray.

  11. Why the transcriptomic analyses may not have revealed all proteins ? • lack of correlation between transcript and disease-associated protein levels • translocation of a protein in the disease state rather than simply differential levels of the transcript • novel/uncharacterized genes that are not highly represented within the "closed system" of a cDNA array

  12. Individual proteins Complete sequence analysis Emphasis on structure and function Structural biology Complex mixtures Partial sequence analysis Emphasis on identification by database matching Systems biology Protein chemistryProteomics

  13. Omics Genome “Genomics” DNA Transcriptome “Transcriptomics” mRNA Proteome “Proteomics” Proteins Metabolome “Metabolomics” Metabolyte

  14. Generalized proteomics scheme Yarmush & Jayaraman, 2002

  15. Definitions of Proteomics • First coined in1995 • Be defined as the large-scale characterization of theentire proteincomplement of a cell line, tissue, or organism. • Goal: -To obtain a moreglobal and integratedview of biology by studying all the proteins of a cell rather than each one individually.

  16. How Proteomics Can Help Drug Development http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18

  17. Why is Proteomics necessary? • Having complete sequences of genome is not sufficient to elucidate biological function. • A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival • Modifications of proteins can be determined only by proteomic methodologies • It is necessary to determine the protein expression level • The localization of gene products can be determined experimentally • Protein-protein interactions • Proteins are direct drug targets.

  18. The Major Techniques in Current Proteomics • Two-dimensional electrophoresis • IEF strip separation • SDS-PAGE gel separation • Mass Spectrometry • Protein sequencing • Peptide mapping • Post-translational Modification • Others • ICAT • Yeast two hybrid assay • Protein chips

  19. Two-dimensional Gel Approach Nature 2000, 405, 837-846

  20. Ionization State as a Function of pH

  21. Two-dimensional Gel Electrophoresis First dimension: IEF (based on isoelectric point) - + Sample acidic basic High MW SDS-PAGE (based on molecular weight) Low MW

  22. Staining of Polyacrylamide Gels Silver staining Coomassie blue staining Sypro Ruby staining

  23. Image Analysis

  24. In-gel digestion-trypsin, chymotrypsin, Glu C, Lys C, Asp N,…

  25. 1. Cut protein spot 2. Protein digestion Protease 4. Spot onto MALDI chip 3. Peptide purification 6. Peptide fragment fingerprint 5. MALDI-TOF analysis Protein Identification by MALDI-TOF

  26. * Trypsin * * * * * * Mass Spectrometric Identification of Proteins - Mapping Peptide mass fingerprinting (PMF) or peptide mapping

  27. Typical mass spectrometry scheme peptide mass fingerprint & tandem mass spectrometry Yarmush & Jayaraman, 2002

  28. How Does a Mass Spectrometer Work? Analyzer Sample input Detector Ionization

  29. The basic components of a mass spectrometry system Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc. Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization (MALDI) etc Analysis: Quadrupole, Time of Flight(TOF), ion trap etc. Detection: Electron multiplier, Scintillation counter

  30. MALDI Mass Spectrometer

  31. Time of flight Mass Spectrometry

  32. Ionization Matrix-Assisted Laser Desorption/Ionization(MALDI) Matrix: - organic acids - benzoic acids

  33. Proteome chip ‘proteome chip’composed of 6,566 protein samples representing 5,800 unique proteins, which are spotted in duplicate on a single nickelcoated glass microscope slide39. The immobilized GST fusion proteins were detected using a labeled antibody against GST. (MacBeath G. Nat Genet 2002 Dec;32 Suppl 2:526-32 )

  34. Clinical and Biomedical Applications of Proteomics • An approach complementary to genomics is required in clinical situations • The clinical applications of 2-D PAGE & MS • Identifying the origin of body fluid samples or the origin of a tissue biopsy. • Analyzing protein phenotypes and protein post-translational modifications in fluid, cells, or tissues. • Examining the clonality of immunoglobulins and detecting clones which are not seen with conventional techniques. • Monitoring disease processes and protein expression. • Discovering new disease markers and/or patterns in body fluids, cells, or tissues.

  35. Body fluids Blood cell Plasma and serum Urine Cerebrospinal fluid Amniotic fluid Synovial fluid Saliva Sweat Tears Semen Solid tissue Heart Brain Thyroid Muscle Malignant diseases Tissue culture Malignant cells Bacterial proteins Clinical applications of 2-D electrophoresis Young & Tracy Journal of Chromatography A, 698 (1995) 163-179

  36. Serial dichromatic detection of glycosylated and un-glycosylated proteins Total proteins Glycoproteins SYPRO Ruby protein gel stain Pro-Q Emerald 488 glycoprotein stain kit

  37. Protein-protein interactions • Protein-protein interactions are intrinsic to every cellular process. • Form the basis of phenomena • DNA replication and transcription • Metabolism • Signal transduction • Cell cycle control • Secretion

  38. The Study of Protein-protein Interaction by Mass Spectrometry bait ? SDS- PAGE ? S14 ? ? * * * * MASS

  39. Yeast Two-hybrid System • Useful in the study of various interactions • The technology was originally developed during the late 1980's in the laboratory Dr. Stanley Fields (Fields and Song, 1989, Nature).

  40. http://us.expasy.org/tools/

  41. Major Directions in Coming Proteomics • Protein structure prediction and modeling • Assignment of protein structure to genomes • Classifications of protein structures • Drug discovery and development

  42. Types of Proteomics and Their Applications to Biology

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