Introduction

1. Introduction 阮雪芬 National Taipei University of Technology Feb 24, 2003

2. Outline • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • Major Directions in Coming Proteomics

3. Outline • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • Major Directions in Coming Proteomics

4. What Is Proteomics ?

5. Proteomics • Protein +GenomeProteome • ProteomeProteomics

6. Outline • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • Major Directions in Coming Proteomics

7. 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.

8. Definitions of Proteomics • 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

9. Proteomics Origins • In1975, the introduction of the 2D gel byO’Farrellwho began mapping proteins from E. coli. • The first major technology to emerge for the identification of proteins wasthe sequencing of proteinsby Edman degradationpicomole • MS technologyhas replaced Edman degradation to identify proteinsfemtomole

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

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 • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • 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 Mass Fingerprint Searching in http://www.expasych/tools/peptident.html Current Opinion in Chemical Biology 2000, 4:489–494

18. Ionization State as a Function of pH

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

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

21. Image Analysis

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

24. 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

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

26. How Does a Mass Spectrometer Work? • 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, ion trap etc. • Detection:

27. Ionization Electrospray

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

29. Isotope-coded Affinity Tags (ICAT) Linker Biotin Thiol-reactive end group ICAT consists ofa biotin affinity group, a linker regionthat can incorporate heavy or light atoms , anda thiol-reactive end groupfor linkage to cysteines Avidin chromatography

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

31. 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 )

32. Microarrays for Genomics and Proteomics • DNA microarray are used forgenetic analysisas well as expression analysis at themRNAlevel. • Protein microarrays are used for expression analysis at theproteinlevel and in the expansive field ofinteraction 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 ofbody fluidsand tissuebiopsies. • 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. Outline • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • 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 ? SDS- PAGE ? S14 ? ? * * * * 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-activation domain GAL4 DNA-binding domain Nature, 2000

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

43. 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

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

45. Yeast Protein Linkage Map Data

46. Useful BioWeb

47. Outline • Introduction to proteomics • Definitions of Proteomics • The major techniques in current proteomics • Protein-protein interaction • Major Directions in Coming Proteomics

48. 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)

49. Bioinformatics in Coming Proteomics • Protein structure prediction and modeling • Assignment of protein structure to genomes • Drug discovery and development