1. Session II Today’s Proteomics 台大生技教改暑期課程

2. What’s “proteomics” ? "The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“ Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium. Electrophoresis 16 (1995) 1090-1094 Two MOST related technologies: 1.2-D electrophoresis: separation of complex protein mixtures 2.Mass spectrometry: Identification and structure analysis

3. Today’s topics 1. Introduction to Proteomics 2. Technology of Proteomics 3. Applications of Proteomics

4. 1. Introduction to proteomics

5. Move over Genome…on to Proteomics If the genome is the blueprint of an organism---who reads it? At this point no computer algorithm can solve this –A computer can decode all 6 reading frames of an organism –A computer can compare these.. But then what?

6. Genomic DNA StructureRegulationInformation Computers cannot determine which of these 3 roles DNA play solely based on sequence… (although we would all like to believe they can) Those are what we need to know about protein

7. Definitions of Proteomics Classical - restricted to large scale analysis of gene products involving only proteins Inclusive - combination of protein studies with analyses that have genetic components such as mRNA, genomics, and yeast two-hybrid Don’t forget that the proteome is dynamic, changing to reflect the environment that the cell is in

8. 1 gene = 1protein? 1 gene is no longer equal to one protein In fact, the definition of a gene is debatable..(ORF, promoter, pseudogene, gene product, etc) 1 gene=how many proteins?

9. Why Proteomics?

10. Differential protein expression Scenario 1: can be analyzed by microarray technology DNA RNAProtein Transcription Translation x1 x4 DNA RNAProtein Transcription Translation x3 Stimulus DNA RNAProtein Transcription Translation x3 Stimulus Scenario 2: can be solved by proteomics technology

11. Co- and Post-translational modification Co-translational modified Post-translational modified

12. Why Proteomics? (summary) Annotation of genomes, i.e. functional annotation –Genome + proteome = annotation Protein Function Protein Post-Translational Modification Protein Localization and Compartmentalization Protein-Protein Interactions Protein Expression Studies –Differential gene expression is not the answer

13. Types of Proteomics Protein Expression –Quantitative study of protein expression between samples that differ by some variable Structural Proteomics –Goal is to map out the 3-D structure of proteins and protein complexes Functional Proteomics –To study protein-protein interaction, 3-D structures, cellular localization and PTMS in order to understand the physiological function of the whole set of proteome.

14. 2. Technology of Proteomics

15. Technology of Proteomics Separation of proteins –1DE (convention) –2DE (modern) –Multi-dimensional HPLC (modern) Analysis of proteins –Edman Sequencing (convention) –Mass Spectrometry (modern) Database utilization

16. Technology, Now and then

17. Traditional RNA technique : Northern blotting Isolated RNA Electrophoresis Blotting 1. Estimated time to get results: 2-3days 2. Expressed Gene (mRNA) checked: 1-8 species 3. Accuracy: Low to moderate Probing Developing Labelling on probes !!

18. High-throughput method: Microarray Labelling on sample mRNA as probe cDNA or oligonucleotide spotted on chips 1. Estimated time to get results: 5-7 days 2. Expressed Gene (mRNA) checked: thousands 3. Accuracy: moderate to high data analysis Clustered genes Clustered experiments

19. Traditional Protein technique: peptide sequencing 1.Protein purification: necessary 2.Protein idetified: 1 per purified sample Cut desired band Peptide N terminal sequencing Database searching for homolog

20. High throughput technique: 2D electrophoresis + Mass spectrometry 1.Protein purification: not necessary 2.Protein idetified: up to thousands per unpurified sample

21. In our course, we will focus much on A. 2-DE B. Mass spectrometry

22. Common process for proteomics research

23. 取材自台大微生物生化系莊榮輝教授網頁

24. A. Two-dimensional Electrophoresis 2-DE

25. Major technique in proteomic research: 2-D electrophoresis (separation) Digest to peptide fragment MS analysis 1.First dimension: denaturing isoelectric focusing separation according to the pI 2. Second dimension: SDS electrophoresis (SDS-PAGE) Separation according to the MW Interested spot

26. Run 2-DE, step by step

27. Run 2-DE step by step

28. The principle of IEF The IEF is a very high resolution separation method, and the pI of a protein can be measured.

29. Immobilized pH gradient strips (IPG strips) Introduced by Gorg. A. Introduced by Gorg. A. Ref: Gorg. A (1994), Westermeier (2001) Ref: Gorg. A (1994), Westermeier (2001) Dried gel strips can be stored at -20 to -80 from months to years. Dried gel strips can be stored at -20 to -80 from months to years.

30. IEF sample loading

31. 2-DE instruments, 1st dimension Amersham BiosciencesBio-Rad

32. Run 2-DE step by step

33. 2-DE instruments, 2nd dimension 16 x 16 cm 8 x 10 cm 23 x 20 cm Amersham Biosciences

34. 2-DE instruments, 2nd dimension Bio-Rad

35. Run 2-DE step by step

36. Examples of 2-DE results D Healthy controlPatient Digest to peptide fragment MS analysis

37. B. Mass spectrometry

38. Major technique in proteomic research: Mass Spectrometry (analysis) Ion source Ion separatordetector Ion source: substance to ion gas Mass analysis: according to mass/charge (m/z) Detection: femtomole –attomole (10 -15 – 10 -18 mole)

39. Commonly used Mass Spectrometer in Proteomics MALDI-TOF Matrix Assisted Laser Desorption Ionization Time Of Flight ESI tandem MS (with HPLC, LC tandem MS or LC MS/MS) Electro Spray Ionization MS Quadrupole

40. Commercial available MALDI-Tof Microflex ™, BrukerMALDI micro™, MicromassVoyager DE-PRO™, ABI

41. Principal for MALDI-TOF MASS

43. Two major types of MALDI-TOF

44. Reflectron enhance the resolution

45. Video for MALDI-Tof

46. Video for MALDI-Tof (reflectron)

47. Typical result from MALDI-Tof

48. Peptide fingerprinting with MALDI-TOF

49. ESI Quadrupole MS Nano electrospray: >30 min spray time for 1  L sample Highly charged molecules are selected by ac modulation of transverse fields

50. Quadrupole Mass filter

51. Typical result from ESI Quadrupole MS From Eckerskorn in “Bioanalytik”, Lottspeich and Zorbas (Eds)

52. Triple Quadrupole Mass Spectrometer CID: Collision Induced Dissociation for acquiring Molecular weight and Structural information

53. Q-TOF Mass Spectrometer Collision Cell

54. Nomenclature for CID fragments

55. CID Mass Spectrum

56. Commercial LC/MS/MS HCT plus, Bruker Q-Tof ultima API, MicromassAPI 4000, API

57. Vedio for LC/MS/MS

58. Structure information resolved by LC/MS/MS Peptide sequenc Post translational modifications –Proteolytic processing, truncation Trypsin, Endoproteinase mapping –Acylation Missing of N-terminal peptide –Phosphorylation Differential mapping /phosphatase treatment Fe 3+ -loaded IMAC column –Glycosylation Neuraminidase treatment

59. Mass Spectrometry: Analyzer (I) MALDI-TOF: TYPE: TOF analyzer Sample status: solid phase. PROs: (1) easy (2) fast (3) high-through (4) sensitive. CONs: (1) only fingerprint of protein, no sequence information (2) results is highly dependent on sample quality.

60. Mass Spectrometry: Analyzer (II) (LC) -tandem MS: TYPE: (1) Triple quadrupole (2) Ion trap (3) Q-TOF Sample status: Liquid phase. PROs: (1) de novo sequencing data available. (2) high sensitivity CONs: (1) Lower through put (2) pricey

61. 3. Application of Proteomics

62. Applications of Proteomics 1. Protein Complexes Mining 2. Yeast Two-hybrid system ( in vivo PIP) 3. Phage display system (in vitro PIP) 4. Protein Arrays 5. SELDI protein chips (Ciphergen)

63. 1. Proteome Complex Mining A “functional” proteomics approach A Proteome Mine Example –ATP is immobilized to beads in “protein kinase” conformation –Total protein is mixed the beads and the mixture “washed” –Remaining proteins isolated and identified…protein kinases, and purine dependent metabolic enzymes Immobilize a putative drug to bead and test for a cellular ligand

64. Cell mapping

65. 2. Yeast Two-Hybrid System (in vivo) Reporter Gene Bait Protein Binding Domain Prey Protein Activation Domain Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription. Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs.

66. Yeast 2 hybrid system, contd.

68. 3. Phage display system (in vitro) SCIENCE VOL 298 18 OCTOBER 2002 Phage minor coat protein Biopanning

69. Applications for Phage display system

70. 4. Protein (micro) arrays Another Functional Proteomics Approach Same concept as a DNA Array Has been published in a peer-reviewed journal Too much expectation lies in with.

71. Protein Microarray G. MacBeath and S.L. Schreiber, 2000, Science 289:1760 arrayIT TM Spotting platform and protein microarray

72. What protein microarray can do? 1. Protein / protein interaction 2. Enzyme / substrate interaction (transient) 3. Protein / small molecule interaction 4. Protein / lipid interaction 5. Protein / glycan interaction 6. Protein / Ab interaction Reference: 1. G. MacBeath and S.L. Schreiber, 2000, Science 289:1760 2. H.Zhu et al, 2001 Science 293:2101 3. Ziauddin J and Sabatini DM, 2001 Nature 411:107

73. Protein microarrays (Ab arrays)

74. Face the real world The true spot quality from real experiment

75. 5. SELDI protein chip (Ciphergen) SELDI – surface enhanced laser desorption/ ionization Protein chips

76. SELDI protein chip (Ciphergen), contd.

77. Video for SELDI protein chip

78. SELDI protein chip (Ciphergen), application Representative “ raw ” spectra and “ gel-view ” (grey-scale) of serum from a normal donor, and from patients with either BPH (benign prostate hyperplasia) or prostate cancer (PCA) using the IMAC3-Cu chip chemistry (Virginia Prostate Center).