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Using Proteomics for Biomedical Research

Using Proteomics for Biomedical Research. Proteomics : coined by Wilkins and Williams …. The study of p rote ins as an result from gen omics …………… Ruedi Abersold …defining the mandate of proteomics :

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Using Proteomics for Biomedical Research

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  1. Using Proteomics for Biomedical Research

  2. Proteomics: coined by Wilkins and Williams …. The study of proteins as an result from genomics…………… Ruedi Abersold …defining the mandate of proteomics : “proteomics represents the effort to establish the identities, quantities, structures and biochemical and cellular functions of all proteins in an organism, organ, or organelle, and how these properties vary in space, time and physiological state.”

  3. Examples of Proteomes: ribosomes yeast nucleosomes transcriptional complexes biopsy samples affinity – chemical or biological interactions – IP etc.

  4. 2-D gel rat brain homogenate 1 mg protein load Resolved over 2000 spots pI and MW of protein spots

  5. 2-D gel of an uncharacterized bacteria collected from Yellowstone National Park Mammoth Hotsprings October 2007 (Collaboration with Bruce Fouke, UIUC Geology)

  6. Interpreting 2-D gel images • Image warping • Normalization • Statistical significance • Real spots versus artifacts • Internal standards • Gel to gel variations • Sensitivity “You’ve got one protein missing …” “No, you’ve one extra protein !”

  7. Human Genome • Nucleic Acids • 30,000 genes • DNA is localized in the nucleus, simple extraction ! • Proteins – extraction is always a challenge ! • localization (compartmentalization e.g. nucleus) • solubility, salt, pH

  8. More on protein extraction challenges: • Membrane proteins (hydrophobic) • Glycoproteins • Timing of protein extraction (e.g. cyclins) • Active versus inactive forms • Isozymes • Post-translational modifications (over 300)

  9. Protein abundance in cell varies by 5-6 orders !! Bruker Daltonics

  10. Fractionation • Protein fractionation and purification is a key step • in proteomics studies • Whole animal › organ › tissue › laser captured cell clusters…. • Whole cell extract › nuclear or cytoplasmic extract • Whole brain › hippocampus › post-synaptic membranes • Avoid protein degradation (proteolysis inhibitors) • For studies involving PTMs (e.g. phosphorylation use • phosphatase inhibitors) • Chromatography (ion exchange, HPLC, affinity…) • Avoid inadvertent chemical modifications (carbamate, • formyl or acetyl from acid treatment…….. etc)

  11. Common techniques for proteomics • Fractionation and isolation (extraction, centrifugation, • chromatography) • Classical methods such as Edman Sequecing, amino acid analysis • Electrophoresis • Immunological methods – antibodies pull-down (IP), • ELISA, western blot, immuno-histology, bead-based Luminex assays…… • Structural methods, scattering, microscopy, NMR • Gel based versus chomatography versus mass spec • Spectroscopy (VIS/UV), Mass spectrometry

  12. Fractionation (Agilent method) Agilent

  13. Beckman PF-2D • First dimension - chromatofocusing • Chromatofocusing is a column based chromatography method separation proteins according to their pI (similar to 2-D gel) • Second dimension is RP-HPLC • This method is non-gel based

  14. Functional Specific Protein Stains • Invitrogen (Molecular Probes) • Pro-Q Diamond – phosphorylation • Pro-Q-Emerald – glycosylation • Quantitative, 3 orders • Good sensitivity • Works well with SYPRO-Ruby • Compatible with mass spec

  15. DIGE • Fluorescence Difference in Gel electrophoresis • Protein samples labeled with CyDyes • Cy3 for Sample 1 • Cy5 for Sample 2 • Cy2, normalization, Samples 1 and 2 • All three labeled samples pooled and run in the same gel

  16. Cy3 channel

  17. Cy5 Channel

  18. Channel Cy3 and Cy5 superimposed

  19. Mass spectrometry to Proteomics is like PCR to Genomics 2002 Nobel Prizes in Chemistry Mass spectrometry for macromolecules "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" Koichi Tanaka John B. Fenn

  20. MALDI ToF MS Matrix Assisted Laser Desorption Ionization Time-of-flight mass spectrometer ESI MS Electro-Spray-Ionization mass spectrtometer

  21. Schematic of a modern MALDI-ToF MS using DHB as matrix

  22. Matrix for MALDI ToF • 2,5-dihydroxybenzoic acid (DHB) • α-cyano-4-hydroxy-cinnamic acid • 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) • Specific compounds for glycoprotein etc

  23. Schematic of ESI MS developed by John Fenn (taken from Fenn’s Nobel Prize lecture)

  24. ETD (Electron Transfer Dissociation) • anion reagent – Fluoranthene • fragmentation is superior over CID • essential for difficult peptides with PTM • enables structural analysis of complex carbohydrate

  25. Dual source ESI for LTQ-Orbitrap

  26. Thermo LTQ CID ETD

  27. Waters Q-ToF MS

  28. Waters Q-ToF (Quadrupole time-of-flight mass spectrometer)

  29. Quantitative Proteomics • Relative Quantitation • Absolute Quantitation • Typically the answer is the presence or absence of a certain protein • Expression • Interaction • Modifications

  30. Top Down Proteomics • 2-D Gel electrophoresis (DIGE) • Mass spec based top down measurement (including using traditional fractionation methods)

  31. Middle Down Proteomics • Spear-headed by Neil Kelleher • Uses mild digestion (enzymatic or chemical cleavages) • CNBr – cleaves at methionine • Acetic Acid, Formic Acid - cleaves at aspartic acid • Formylation and acetylation precaution !

  32. Bottom Up Proteomics • Label Free mass spec method • MudPIT • Waters High – Low method • Stable-isotope labeling method • Metabolic • ICAT • iTRAQ • O-18

  33. Absolute Quantitation • Typically using a Triple-Quadrupole MS • Add known amount of specific stable isotope labeled peptide • Also known as Accurate Mass Tagging method (R. Smith)

  34. ICAT

  35. Applied Biosystems Q-Trap 5500 • hybrid triple quad/ion-trap MS • 6 orders of dynamic range • Resolution 3000

  36. Interactome • Cell Research | Vol 18 No 7 | July 2008 • Mapping the human protein interactome • Daniel Figeys - The Ottawa Institute of Systems Biology, The Department of Biochemistry, Microbiology and Immunology, University of Ottawa, • Ottawa, ON, K1H 8M5, Canada • Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other • molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. • Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular • interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but • few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the • developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein

  37. Specific Proteomics Reactor • Daniel Figeys at Ottawa, Canada • Glycomics Reactor • Phosphorylation Reactor • Ubiquitin Reactor • Interactome Reactor

  38. Thermo LTQ-FT-ICR-MS • attomole • sensitivity • widest dynamic • range ( > 4,000) • ppb mass • accuracy • resolution • >750,000 • ECD

  39. Top Down Proteomics • McLafferty (Cornell) • Neil Kelleher (formerly UIUC Chemistry) • Uses FT-ICR-MS • Middle down approach (mild digestion) • Pro-cite program

  40. Thermo-LTQ-Hybrid/Orbitrap

  41. Thermo-Orbitrap

  42. Waters Q-ToF SYNAPT G2 • Newest member of Waters high resolution Q-ToF • family of mass spectrometers • First commercial Mass Spectrometer capable of measuring ion-mobility • Uses T-Wave (traveling wave) to improve ion mobility • T-Wave uses RF • Separation by mass, charge and shape

  43. Using Ion Mobility MS to differentiate neuropeptides differing by one D/L amino acid • YdAEFL amide • YlAWFL amide • FdMRF amide • FlMRF amide

  44. Comparison study between • Thermo LTQ, • Thermo LTQ-Orbitrap • Waters SYNAPT

  45. (Slide taken from Bruker Daltonics)

  46. Jonathan Sweedler Laboratory (UIUC) Micro-sampling technique • Sampling of neuropeptides using SPE beads • Aplysia – abdominal bag cell clusters • PAC (Pleural Abdominal Connective) nerve • stimulation • Beads placed before, during PAC stimulation • Neuropeptides varies many orders, verified by MS

  47. AA • Images obtained from a • mouse kidney section. • digital photograph • total ion count • Ion 4,965 Da • Ion 11,362 Da MALDI Imaging (taken from Waters literature)

  48. Proteomics - Challenges • Sample • Sampling Handling • Complexity Reduction • Chromatography • Mass Spectrometry • Bioinformatics (Abersold (2009) Nature Methods, vol 6, 411)

  49. Proteomics Center Carver Biotechnology Center

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