1. Proteomics and Bioinformatics 阮雪芬 NTU Dec 25, 2002

2. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

3. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

4. What Is Proteomics ?

5. Proteomics Protein +Genome  Proteome Proteome  Proteomics

6. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

7. Definitions of Proteomics First coined in 1995 Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. 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. The classical definition Two-dimensional gels of cell lysate and annotation. Two-dimensional gels to visualize differential protein expression. In the post-genomics era Protein Identification Post-translational modifications Determining Function Molecular Medicine Differential display by two-dimensional gels Protein-Protein Interactions Definitions of Proteomics

9. 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

10. How Proteomics Can Help Drug Development

12. 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.

13. Types of Proteomics and Their Applications to Biology

14. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

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

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

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

18. Ionization State as a Function of pH

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

20. Silver staining Coomassie blue stainingSypro Ruby staining Staining of Polyacrylamide Gels

21. Image Analysis

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

24. 1. Cut protein spot2. Protein digestion 3. Peptide purification4. Spot onto MALDI chip 5. MALDI-TOF analysis 6. Peptide fragment fingerprint Protease Protein Identification by MALDI-TOF

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

26. Sample Input: Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc. Ionization: Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization (MALDI) etc Analysis: Analysis: quadrupole, time of flight, ion trap etc. Detection: Detection: How Does a Mass Spectrometer Work?

27. Electrospray Ionization

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

29. Isotope-coded Affinity Tags (ICAT) Avidin chromatography Biotin Linker Thiol- reactive end group ICAT consists of a biotin affinity group, a linker region that can incorporate heavy or light atoms, and a thiol-reactive end group for linkage to cysteines

30. NATURE, VOL 405, 15 JUNE 2000 A strategy for mass spectrometric identification of proteins and post- translational modifications

31. ‘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 ) Proteome chip

32. Microarrays for Genomics and Proteomics DNA microarray are used for genetic analysis as well as expression analysis at the mRNA level. Protein microarrays are used for expression analysis at the protein level and in the expansive field of interaction analysis.

33. Protein Microarrays In Medical Research Accelerate immune diagnostics. The reduction of sample volume----the analysis of multiple tumor markers from a minimun amount of biopsy material. New possibilities for patient monitoring during disease treatment and therapy will be develpoed based on this emerging technology.

34. Clinical and Biomedical Applications of Proteomics An approach complementary to genomics is required in clinical situations to better understand epigenetic regulation and get closer to a "holisitic" medical approach. The potential clinical applications of 2-D PAGE, especially to the analysis of body fluids and tissue biopsies. 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. Clinical applications of 2-D electrophoresis 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 Young & Tracy Journal of Chromatography A, 698 (1995) 163-179

36. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

37. Protein-protein Interaction Introduction Mass Spectrometry Yeast Two-hybrid Assay

38. 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

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

40. 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).

41. Yeast Two-hybrid System GAL4 DNA- binding domain GAL4 DNA- activation domain Nature, 2000

42. Yeast Two-hybrid System Library-based yeast two-hybrid screening method Nature, 2000

43. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

44. 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/Vidal/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html

45. 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

46. Yeast Protein Linkage Map Data

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

48. GeneScape

50. GeneScapeMIPS Currently about 9750 protein-protein-interactions (8250 physical and 1500 genetic) are annotated.

51. Yeast Interacting Proteins Database (YIPD) http://genome.c.kanazawa-u.ac.jp/ Yeast Interacting Proteins Database

52. Genetic Network Visualization System Workbench System for Support of Gene Regulatory Network Construction YIPD

53. Java Applet YIPD

54. GUI System Help YIPD

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

56. Pathway Software BIOCARTA

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

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

59. Useful BioWeb 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

60. Outline Proteomics Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Bioinformatics Protein-protein Interactions Useful Bio-websites Major Directions in Coming Proteomics

61. Chemical proteomics (screens for activity and binding) Structural proteomics (target validation and development) Interaction proteomics (identification of new protein targets) Bioinformatics (annotation of the proteome)

62. Major Directions in Coming Proteomics Protein stability and folding Applications of hydrophobicity Superposition of structures, and structural alignments DALI (Distance-matrix alignment) Evolution of protein structures Classifications of protein structures

63. Major Directions in Coming Proteomics Protein structure prediction and modeling Assignment of protein structure to genomes Prediction of protein function orthologues and paralogues Drug discovery and development