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Today’s Proteomics

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Today’s Proteomics

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  1. Today’s Proteomics Session II 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: • 2-D electrophoresis: separation of complex protein mixtures • Mass spectrometry: Identification and structure analysis

  3. Today’s topics • Introduction to Proteomics • Technology of Proteomics • 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 • Structure • Regulation • Information • 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 Stimulus Stimulus RNA Protein DNA Transcription Translation x4 x1 Scenario 1: can be analyzed by microarray technology RNA Protein DNA Transcription Translation x3 Scenario 2: can be solved by proteomics technology RNA Protein DNA Transcription Translation x3

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

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

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

  20. High throughput technique: 2D electrophoresis + Mass spectrometry • Protein purification: not necessary • 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) • First dimension: • denaturing isoelectric focusing • separation according to the pI • 2. Second dimension: • SDS electrophoresis (SDS-PAGE) • Separation according to the MW Interested spot MS analysis Digest to peptide fragment

  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. • Ref: Gorg. A (1994), Westermeier (2001) • 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 Biosciences Bio-Rad

  32. Run 2-DE step by step

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

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

  35. Run 2-DE step by step

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

  37. B. Mass spectrometry

  38. Major technique in proteomic research:Mass Spectrometry (analysis) Ion source Ion separator detector 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 ESItandem MS (with HPLC, LC tandem MS or LC MS/MS) • Electro Spray Ionization MS Quadrupole

  40. Commercial available MALDI-Tof Microflex ™, Bruker Voyager DE-PRO™, ABI MALDI micro™, Micromass

  41. Principal for MALDI-TOF MASS

  42. 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 Gel Database Protein ? 1 2 3 tryptic stored data or theoretical? peptides digestion mass spectrometry compare: ?? is identical to ??

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

  50. Quadrupole Mass filter