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Proteomic Applications

Proteomic Applications. Isabelle Noël-Georis Lab. Biological Chemistry UMH, Mons Tel 065/373312 Fax 065373320 Email isabelle.georis@umh.ac.be. Key steps in proteomic analysis The standard : 2D and mass spectrometry 3. Alternatives : 3.1. SDS-PAGE and mass spectrometry

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Proteomic Applications

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  1. Proteomic Applications Isabelle Noël-Georis Lab. Biological Chemistry UMH, Mons Tel 065/373312 Fax 065373320 Email isabelle.georis@umh.ac.be

  2. Key steps in proteomic analysis • The standard : 2D and mass spectrometry • 3. Alternatives : • 3.1. SDS-PAGE and mass spectrometry • 3.2. Chromatography and mass spectrometry • 3.3. Protein arrays • Hot topics • 4.1. Peptidomics • 4.2. SELDI • 4.3. Interaction Proteomics

  3. Technologies for Proteomics Isolation As many proteins as possible Disruption of aggregates Reproducibility Inhibit proteases 2-DE Separation Highest resolution Reproducibility Lowest bias against any type of protein Detection Highest sensitivity Widest dynamic range Compatible with further analyses Identification Quantification Bioinformatics Edman Mass Spectrometry

  4. 2D gels-based proteomics : a case study 1. Protein Separation

  5. 2D gels-based proteomics : a case study 2. Protein Quantification

  6. 2D gels-based proteomics : a case study 3. Protein Identification

  7. 2-DE : Pro’s and Con’s + - Answers Reproducibility Resolution Quantification Isoform display High discovery potential Low abundance proteins Low molecular weight proteins Less soluble proteins Proteins with extreme pI Difficult automatization Prefractionation narrow range pH gradients Prefractionation Optimized solubilization Alkaline and acidic pH gradients And … gel free-based techniques for protein separation

  8. Key steps in proteomic analysis • The standard : 2D and mass spectrometry • 3. Alternatives to 2-DE • 3.1. SDS-PAGE and mass spectrometry • 3.2. Multidimensional liquid chromatography • 3.3. ICAT technology • 3.4. Protein arrays • 3.5. Peptidomics • 3.6. SELDI • Hot topics • 4.1. Interaction Proteomics • 4.2. Immunoproteomics

  9. Alternatives to 2-DE

  10. SDS-Page followed by MS 1. Low complexity sample (Organite, complex, subfraction …) 1 band = 1 protein species Sufficient resolution, possible quantification + Membrane Proteins Extreme pI - Resolution Isoforms 2. High complexity sample 1 band = numerous protein species Unsufficient resolution, impossible quantification Output = list of protein

  11. Multidimensional liquid chromatography + Membrane Proteins Extreme pI, MW Sensitivity Automatisation - Isoforms Quantification Link et al. Nat. Biotechnol. 1999 Washburn et al. Nat. Biotechnol. 2001

  12. ICAT Technology - Isoforms Intra-sample quantification + Membrane Proteins Extreme pI, MW Sensitivity Automatisation Inter-sample quantification Gygi et al. Nat. Biotechnol. 1999 Han et al. Nat. Biotechnol. 2001

  13. Complex biological sample 1! molecule … * * * G A B C D E F M H I J K L N O P a-B a-K a-A a-L a-C a-D a-J a-F a-G a-H a-I a-E ProteinX a-ProteinX GST … Zhu et al., Science 2001 Haab et al., Genome Biology 2001 Protein arrays + Extreme pI, MW Sensitivity Automatisation Quantification Resolution Protein function arrays Protein-detecting arrays (Kinase activity, interaction with DNA, proteins, lipids …) - Membrane proteins Isoforms Antibody availability Cross-reactivity Ligand activity Information on the protein Indirect detection

  14. Peptidomics Schrader et al. Trends Biotechnol. 2001

  15. SELDI (Surface Enhanced Laser Desorption Ionization) APPLICATIONS Protein Profiling/ Classical Proteomics • Protein Profiling/Differential Protein Expression • Toxicology – marker discovery and validation • Clinical Diagnostics – marker discovery and validation • Disease monitoring (drug efficacy studies) • Pharmacokinetics Protein-Protein Interactions • Immunoassay development and screening • Receptor-ligand assays • Protein-protein interactions • DNA-protein interactions Protein Purification and Characterization • Purification development and monitoring • Peptide Mapping • Sequencing • PT modification analysis (phosphorylation, glycosylation) • Epitope Mapping

  16. Hot Topics

  17. Tandem Affinity Purification

  18. Tandem Affinity Purification (Gavin et al., Nature 2002)

  19. Worm Interactome version 5

  20. Worm Interactome version 5 ) 4027 2135 1892 108 (10) 949 5534 interactions, 15% of the C. elegans proteome connected

  21. Immunoproteomics : primary reaction of the host

  22. Immunoproteomics : primary reaction of the host

  23. Immunoproteomics : primary reaction of the host

  24. Immunoproteomics : humoral reaction of the host

  25. Immunoproteomics : humoral reaction of the host

  26. Immunoproteomics : humoral reaction of the host

  27. Immunoproteomics : cellular reaction of the host

  28. Immunoproteomics : cellular reaction of the host

  29. Immunoproteomics : cellular reaction of the host

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