1 / 78

Today’s Proteomics

Today’s Proteomics. Session II. 台大生技教改暑期課程. What’s “proteomics” ?. "The analysis of the entire prote in complement expressed by a gen ome , or by a cell or tissue type.“

nusa
Download Presentation

Today’s Proteomics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related