1. 蛋白質體學 阮雪芬 Jul 18 & 25, 2003

2. Outline The characters of proteins Differences between protein chemistry & proteomics Why to study proteome Proteomics Introduction to proteomics Definitions of proteomics The major techniques in current proteomics Protein-protein interactions

3. The characters of proteins

4. DNA 和蛋白質合成的地方

5. 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.

6. Why to study proteome ?

7. 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

8. 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 chemistry Proteomics

9. Introduction To Proteomics

10. DNA mRNA Proteins Cell functions Genome “ Genomics ” Proteome “ Proteomics ” Genomics vs. Proteomics

11. Generalized Proteomics Scheme Yarmush & Jayaraman, 2002

12. Definitions of Proteomics

13. 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.

14. 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

15. 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

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.

19. Jürgen Drews, 2000

20. 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

21. 各項產品營業收入 資料來源： Amgen, Inc.

22. The Major Techniques in Current Proteomics

23. Two-dimensional electrophoresis IEF strip separation SDS-PAGE gel separation Mass Spectrometry Protein sequencing Peptide mapping Others ICAT Yeast two hybrid assay Protein chips

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

25. 3.510 42 60 70 150 kDa pH Increase of 50% Decrease of 50% Unmatched spots Matched spots Image Matching

26. www.expasy.ch/ch2d

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

30. 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

31. Yarmush & Jayaraman, 2002 Typical mass spectrometry scheme peptide mass mapping and tandem mass spectrometry

32. Ionization State as a Function of pH

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

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

35. Image Analysis

36. In-gel Digestion Enzyme: trypsin chymotrypsin

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

38. 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

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

40. 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: How Does a Mass Spectrometer Work?

41. Electrospray Ionization

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

43. 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

44. NATURE, VOL 405, 15 JUNE 2000 A Strategy for Mass Spectrometric Identification of Proteins and Post- translational Modifications

45. ‘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

46. 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.

47. 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.

48. 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.

49. 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

50. Protein-protein Interactions

51. Introduction Mass Spectrometry Yeast Two-hybrid Assay

52. 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

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

54. 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).

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

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

57. 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

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

59. Pathway Software BIOCARTA

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

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

62. 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

63. Major Directions in Coming Proteomics 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)

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

65. Types of Proteomics and Their Applications to Biology

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

67. Thank You!