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

17. Standard Proteome Analysis by 2DE-MS
Mass Fingerprint Searching in
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
(based on molecular weight)
SDS-PAGE
Low MW

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

21. Image Analysis

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

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

25. How Does a Mass Spectrometer Work?
Sample input
Analyzer
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. 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 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
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: )

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)

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
C. Elegans
H. Pylori
Drosophila

44. Yeast Protein Linkage Map Data
New protein-protein interactions in yeast
List of interactions with links to YPD
Stanley Fields Lab

45. Yeast Protein Linkage Map Data

46. Information available
Useful BioWeb
Site name
URL
Information available
MOWSE
Peptide mass mapping and sequencing
ProFound
PeptIdent
PepSea
MASCOT
PepFrag
Protein Prospector
FindMod
Posttranslational modification
SEAQUEST
Uninterpreted MS/MS searching
FASTA Search Programs
Protein and nucleotide database searching
Cleaved Radioactivity of
Phosphopeptides
Protein phosphorylation site mapping

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