1. Yuki Juan ( 阮雪芬 ) Aug 7, 2003 Bioinformatics Data Analysis in Proteomics

2. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

3. Proteomics Network Identify Proteins Drug Discovery Structures Protein-Protein Interactions Pathways Protein Functions

4. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

5. What Is Proteomes ?

6. Proteomes Gene + Chromosome  Genome Protein +Genome  Proteome Proteomes are dynamics Proteome changes as a function of: time development extracellular condition intracellular condition

7. Definitions of Proteomics First coined in 1995 by Wilkins Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. The study of proteomes Goal: -To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually.

8. Proteomics Origins In 1975, the introduction of the 2D gel by O’Farrell who began mapping proteins from E. coli. The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradation  picomole MS technology has replaced Edman degradation to identify proteins  femtomole

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

10. 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.

11. Types of Proteomics and Their Applications to Biology

12. Mechanisms by Which a Single Gene Can Give Rise to Multiple Gene Products In bacteria, 1 or 2 proteins/gene In yeast, 3 proteins/gene In human, 3 or more proteins/gene Glycosylation

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

14. 3.510 42 60 70 150 kDa pH Increase of 50% Decrease of 50% Unmatched spots Matched spots Image Matching

15. www.expasy.ch/ch2d

17. http://www.expasy.ch/melanie/

19. Standard Proteome Analysis by 2DE-MS Current Opinion in Chemical Biology 2000, 4:489–494 Mass Fingerprint Searching in http://www.expas y.ch/tools/peptide nt.html

20. ControlTreated sample Example: Gel

21. + - gel membrane Transfer proteins from gel to PVDF membrane Sequencing (Very time-Consuming)

22. Mass spectrometry

23. For peptide mass fingerprint data

24. Species pI and MW

25. Peptide Mass Fingerprinting

26. World Wide Web Tools for Searching Databases Site nameURLInformation available MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowsePeptide mass mapping and sequencing ProFoundhttp://prowl.rockefeller.edu/cgi- bin/ProFound Peptide mass mapping and sequencing PeptIdenthttp://www.expasy.ch/tools/peptident.Peptide mass mapping and sequencing PepSeahttp://195.41.108.38/PepSeaIntro.htmlPeptide mass mapping and sequencing MASCOThttp://www.matrixscience.com/Peptide mass mapping and sequencing PepFraghttp://www.proteometrics.com/Peptide mass mapping and sequencing Protein Prospectorhttp://prospector.ucsf.edu/Peptide mass mapping and sequencing FindModhttp://www.expasy.ch/tools/findmod/Posttranslational modification SEAQUESThttp://fields.scripps.edu/sequest/Uninterpreted MS/MS searching FASTA Search Programs http://fasta.bioch.virginia.edu/Protein and nucleotide database searching Cleaved Radioactivity of Phosphopeptides http://fasta.bioch.virginia.edu/crpProtein phosphorylation site mapping

27. The Information Stored in Genes Is Expressed by a Multistage Process

28. DNA 和蛋白質合成的地方 DNA Proteins Sugar Chain

29. Post-translational Modification Genomics provides comprehensive data-bases of sequence information, DNA and mRNA provide no information concerning the activities and post- translational modifications of proteins. The number of documented protein co- and post- translational modifications now exceeds 400 (http: / / abrf.org / index-.cfm/dm.home). The elucidation of protein post-translational modifications is perhaps the most important justification for proteomics as a scientific endeavor.

30. Chemical modification Covalent linkage of chemical group to amino acid in protein Several types: Acetylation (common on first amino acid) Phosphorylation (often involved in regulation) Lipidation (attachment to membrane) Glycosylation (common outside cell)

31. Processing Removal of part of protein by either: Cleavage (protease cuts protein into smaller pieces) Splicing (self-removal and rejoining of ends)

33. Post-translational Modification

34. Glycosylation Mass spectrometric “sequencing” of oligosaccharides Increase the complexity of the proteome

35. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

36. Protein Functions Gene Ontology http://www.geneontology.org/ GoMiner http://discover.nci.nih.gov/gominer

37. Gene Ontology

39. GoMiner

40. Genome Biology 2003, 4:R28

41. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

42. Protein-protein Interactions Introduction Mass Spectrometry Yeast Two-hybrid Assay

43. Introduction Kanehisa (2000)said: Interaction NetworkFunction Post-genome informatics (2000)

44. Introduction 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

45. Introduction Knowledge of interacting proteins Provide insight into the function of important genes Elucidates relevant pathways Facilitates the identification of potential drug targets Use in developing novel therapeutics

46. Interaction Discovery Methods TiBS(2003), 27(12), 633-638.

47. Structural Biology of Interactions and Complexes X-ray crystallography Nuclear Magnetic Resonance (NMR) Electron microscopy (EI)

48. Examples of Three-dimensional Protein Interactions and Complexes TiBS(2003), 27(12), 633-638.

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

50. Peptide Mass Fingerprinting

51. Detecting Protein-protein Interactions by Protein Microarray

52. 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 (see Fields and Song, 1989, Nature).

53. Yeast Two-hybrid Assay GAL4 DNA- binding domain GAL4 DNA- activation domain Nature, 2000

54. Yeast Two-hybrid Assay Library-based yeast two-hybrid screening method Nature, 2000

55. Information Extraction (IE) A vast amount of data on protein- protein interactions residues in the published literature, which never been entered into databases. IE have been applied to gaining information on protein-protein interactions.

56. Mining Literature for Protein-protein Interactions

59. Protein-protein Interactions on the Web Yeast http://depts.washington.edu/sfields/yplm/data/index.html http://portal.curagen.com http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://www.pnas.org/cgi/content/full/97/3/1143/DC1 http://dip.doe-mbi.ucla.edu/ http://genome.c.kanazawa-u.ac.jp/Y2H C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vid al/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html

60. Yeast Protein Linkage Map Data New protein-protein interactions in yeast Stanley Fields Lab http://depts.washington.edu/sfields/yplm/data List of interactions with links to YPD

61. Yeast Protein Linkage Map Data

62. GeneScape PathwayCalling: Protein interaction and pathway Analysis http://portal.curagen.com PATHCALLING YEAST DATABASE

63. GeneScape

64. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

65. Biological Pathways

66. Metabolic and biochemical Transcription, regulation and protein synthesis Signal transduction

67. Metabolic and Biochemical Pathway The synthesis of fatty acids

68. Transcription, Regulation and Protein Synthesis Transcription Initiation Elongation Termination

69. Initiation of Transcription

70. The Elongation of Transcrption

71. The Termination of Transcription

72. Transcription, Regulation and Protein Synthesis Translation Initiation Elongation Termination

73. 30S Subunit and Shine-Dalgarno Sequence IF: Initiating protein factor E: Exit site P: Peptide site A: Aminoacyl site

74. Initiation of Translation in E. coli

75. The Elongation Process in Protein Synthesis EF-Tu: Elongation Factor Peptide transferase

76. Termination of Protein Synthesis Stop codon: UAG, UAA, UGA Cytoplasmic protein release factor

77. Signal Transduction EDF signaling pathway Protein kinase

78. Pathway Conservation

79. Databases of Pathways---KEGG http://www.genome.ad.jp/kegg/

80. Glycolysis

81. Pathway Software BIOCARTA http://biocarta.com/ Browse all pathway

82. Pathway Software BIOCARTA

83. Pathway Result 1: Enolase Glycolysis Pyruvate Acetyl-CoA ethanol lactate Cancer cells BIOCARTA

84. Pathway Result 2 : Retinoic Acid Receptor RXR-alpha BIOCARTA

85. Development of the Network- based Pathway Paradigm Ti BS (2003), 28(5), 250-258

86. Development of the Network- based Pathway Paradigm Ti BS (2003), 28(5), 250-258

87. BIND http://bind.mshri.on.ca/

88. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

90. Protein Structure Data Bank- PDB http://www.rcsb.org/pdb/

91. Therapeutic Target Classes

92. Amgen （ Applied Molecular Genetics ） 成立日期： 1980 年 4 月 8 日 CEO ： Kevin W. Sharer 員工人數： 6342 市場總值： 698.4 億美元 產品項目：重組蛋白藥物 EPOGENR (Epoetin alfa) NEUPOGENR (Filgrastim) INFERGENR (Interferon alfacon-1) 資料來源：彭博資訊社、 Zacks.com ， 6/14/2001

93. 各項產品營業收入 資料來源： Amgen, Inc.

94. Examples of Structure-based Drug Design HIV protease with the inhibitor amprenavir (Agenerase) bound Close-up of zanamivir (Relenza) bound to influenza neuraminidase

95. Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

96. Useful Bio Websites PROSITE http://tw.expasy.org/prosite/ Amos' WWW links page http://tw.expasy.org/alinks.html Phosphoprotein Database (PPDB) http://www- lmmb.ncifcrf.gov/phosphoDB/

97. PROSITE is a database of protein families and domains. http://tw.expasy.org/prosite/

99. http://tw.expasy.org/alinks.html

100. http://tw.expasy.org/alinks.html#PTM

101. http://www.cbs.dtu.dk/databases/PhosphoBase/

104. Proteomics Network Identify Proteins Drug Discovery Structures Protein-Protein Interactions Pathways Protein Functions