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Proteomics

Proteomics. What is it? How is it done? Are there different kinds? Why would you want to do it (what can it tell you)?. The Central Dogma of Molecular Biology. DNA. transcription. RNA. translation. Protein. Key Concept: Proteins do the work everything else is mostly information.

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Proteomics

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  1. Proteomics What is it? How is it done? Are there different kinds? Why would you want to do it (what can it tell you)?

  2. The Central Dogma of Molecular Biology DNA transcription RNA translation Protein Key Concept: Proteins do the work everything else is mostly information Key Concept: Proteomics is the “high throughput” analysis of proteins.

  3. DNA Genomics RNA Genomics/Transcriptomics Protein Proteomics Metabolites Metabolomics The Central Dogma-omics

  4. DNA Genomics ~6-45,000 parts RNA Genomics/Transcriptomics ~100,000 parts Protein Proteomics ~1,000,000 parts Metabolites Metabolomics >500,000,000 parts The Central Dogma-omics Key Concept: Complexity increases the farther away from information you get Key Concept: Chemical complexity also increases the farther away you get

  5. Protein Machines The polyAdenylation Machinery The Proteosome Key Concept: Biochemical functions are carried out by multi-protein machines Key Concept: A Protein Function can be inferred by it’s binding partners Key Concept: Knowledge of a Machine’s components is required to understand how it works and how it is regulated

  6. Protein Networks 2 Steps Key Concept: Protein Machines are organized into larger Networks

  7. Proteins are Organized in Super Networks Key Concept: The proteome is HIGHLY Networked

  8. Key Concept: Proteins vary widely in concentration

  9. Major Types of Proteomics Interactomics: Mapping Protein:Protein Interactions -Yeast 2-hybrid techniques -high throughput protein identification by Mass Spectrometry Survey Proteomics: Qualitative or Quantitative Analysis of the protein component -whole organism, tissue, cell type, or subcellular compartment -2D gel electrophoresis ->MS -typically a few 100 proteins -Multidimensional LC->MS/MS -typically a few 1000 proteins Identification of Biomarkers

  10. Information Trade Offs Vida infra G. MacBeath

  11. Proteins are Organized in Super Networks Key Concept: The proteome is HIGHLY Networked

  12. Time of Flight Mass Spectrometer Timed ion selector Laser Reflector Sample plate + + + + + + + Flight tube Accelerating field Detectors Key Concept: Mass Spectrometers can only measure charged species. Key Concept: All Mass Spectrometers have at least 2 parts: an ion source and a mass analyzer

  13. A Quadrupole Mass Filter

  14. Ion Trap Mass Spectrometer Key Concept: Mass Spectrometers can only measure ions and more accurately, the behavior of ions in an electric field

  15. A Generic Mass Spectrum Key Concept: A mass spectrum is a graphical representation of the the data, with intensity on the y axis and m/z on the x axis

  16. Important Properties of a Mass Spectrum Mass is expressed as m/z (mass/charge). m=1000 z=1 m/z =1001 m=1000 z=2 m/z =501 m=1000 z=3 m/z =334.3 Intensity is usually normalized to 100 Intensity can be expressed as ion current or ion counts The intensity is related to abundance, but only when you are referring to the same compound. Mass accuracy is an important parameter that affects downstream analysis. Mass accuracy is usually expressed as ppm. EM=1000.1 TM=1000 ppm= (0.1/1000) x 106 100 ppm

  17. How to Identify Proteins Proteolysis Proteolysis Proteolysis Key Concept: Each unique protein will give rise to a unique set of peptides

  18. Proteolysis Peptide Mass Fingerprinting Key Concept: A single protein yields many proteolitic fragments

  19. Proteolysis Peptide Mass Fingerprinting Key Concept: A mass spectrometer is a very accurate scale!

  20. Peptide Mass Fingerprinting

  21. Peptide Mass Fingerprinting

  22. Peptide Mass Fingerprinting

  23. Peptide Mass Fingerprinting

  24. Peptide Mass Fingerprinting Key Concept: Each mass pulls out ~11,000 candidate proteins

  25. Peptide Mass Fingerprinting Key Concept: Ideally only one protein should be uniquely identified

  26. Key Concept: Fragments can be matched to a database of proteins. The number of indentified proteins is related to the mass accuracy.

  27. Peptide Mass Fingerprints often FAIL to give Significant Results -Protein Mixtures “confuse” the statistical algorithms -Not enough high quality peaks -Too many “popular masses” -Possible to “play” with the search parameters to give a statistically meaningful result. Can be overcome by performing peptide fragmentation (AKA MS/MS)

  28. Thermo Demo Movie

  29. Proteolysis What is MS/MS -MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.

  30. What is MS/MS -MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.

  31. How do you interpret an MS/MS spectra?? A M + A S R R E M P S E L P L A M R S P + E L R A E M + S L P R + A E P M S L

  32. A M R S P + E L

  33. How do you interpret an MS/MS spectra?? • Steps for Manual Interpretation • Label the precursor masses • Label any obvious water losses • (m/z with D18) • Look for any peaks with D28 • (a – b ion pairs) • Look up the mass of the first b ion • in the dipeptide chart. • Begin building the b ion series and • use the y ion series for confirmation using • the tables. • Extend till the you reach the presurser mass. • (the last amino acid in the b ion series should • be a Lys or Arg and will be +18 relative to the • mass in the table.)

  34. 99.1 213.1

  35. 99.1 213.1

  36. 99.1 213.1

  37. V 99.1 213.1

  38. V N 114

  39. Q V N 128 114

  40. I/L 113 Q V N 128 114

  41. I/L Q G V N I S E I/L T K

  42. I/L Q I/L N Q V G V N I S E I/L T K

  43. 584.10 826.25 175.00 417

  44. 157.04

  45. Proteolysis Key Concept: A sample analyzed by MALDI can generate between 10-20 MS/MS spectra before it is consumed.

  46. How to Identify Proteins C E Protein Bands are cut from gel, trypsinized and analyzed by mass spectrometry -direct sequencing -search a database (probability based ID) Key Concept: A few MS/MS spectra or even MS/MS spectra from a single protein can be manually interpreted

  47. Proteolysis Proteolysis Proteolysis MS/MS allows for Shot Gun Proteomics Proteolysis Key Concept: Shot Gun Proteomics lets you look at many things at once

  48. Protein ID by Mass Spectrometry

  49. Protein ID by Mass Spectrometry ~10,000 MS/MS per hour Key Concept: LC-MS/MS data is time restricted by the elution profile of the peptides. Maldi is restricted by sample consumption.

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