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  1. Proteomics Astrid Bruckmann IBL, Leiden University Workshop Transcriptomics and Proteomics in Zebrafish, Leiden University,13-22 March 2006

  2. Why Proteomics? Genome - Transcriptome - Proteome from: Graves and Haystead, 2002 • several levels of regulation from gene to function • Proteins are the ultimate operating molecules producing the physiological effect • Proteome: the protein complement of a genome Proteomics: large-scale characterization and functional analysis of the proteins expressed by a genome

  3. Types of proteomics and their application to biology from: Graves and Haystead, 2002

  4. Proteomics - the challenge • The Proteomeis: - dynamic - highly complex - relative protein abundances in a cell can differ from 105 up to about 1010 • Proteomics aims to analyze the levels and structure of all proteins present in a cell or a tissue including their post-translational modifications (Honoré and Østergaard, 2003) • Proteomics approaches include: 1) protein identification 2) protein quantitation or differential analysis 3) protein-protein interactions 4) post-translational modifications 5) structural proteomics Proteomics is complementary to transcriptomics and metabolomics, integration of different -omics data should lead to a more complete understanding of biological systems at a molecular level

  5. Proteomics - the classical definition Two-dimensional gelelectrophoresis (2D-PAGE) of cell lysates Mass spectrometry + - High resolution 2D-PAGE first developed in 1975 (O’Farrell and Klose) - Combination with biological mass spectrometry (1990s) - Availability of genome sequences in databases  central role in proteomic studies generates global patterns of protein expression  annotation  large-scale visualization of differential protein expression Peptide mass fingerprinting for protein identification

  6. First dimension: Isoelectrofocusing (IEF) strip containing a pH gradient immobilized on a gel matrix (Garfinet al. 2000)

  7. 9.5 7.5 9.5 7.5 7.5 pI 4.5 pI 4.5 7.5 9.5 7.5 pI 4.5 pI 4.5 pI 3.5 9.5 9.5 7.5 pI 3.5 7.5 9.5 7.5 7.5 7.5 9.5 9.5 9.5 9.5 Position of proteins before IEF Position of proteins after IEF

  8. Second dimension: SDS-PAGE MW • Proteins enter SDS-Polyacrylamide • gel and are dissolved according to • their molecular mass • Postelectrophoretic staining of the • proteins with: • Coomassie, • Silver, • Fluorescent stains (SYPRO Ruby)

  9. 2D-PAGE based expression proteomics • Protein expression profiling: ~ 1000 proteins routinely detectable in a • 2D-gel  global changes in the proteome readily detectable • posttranscriptional control mechanisms can influence protein expression • posttranslational modifications of a protein such as phosphorylation, glycosylation, processing of signal sequences or degradation can be visualized pI MW SYPRO Ruby stained gel

  10. Protein identification by peptide mass fingerprinting from Graves and Haystead, 2002 • The unknown protein is excised from a gel and converted to peptides by the action of a specific protease. The mass of the peptides produced is then measured in a mass spectrometer. • (B)The mass spectrum of the unknown protein is searched against theoretical mass spectra produced by computer-generated cleavage of proteins in the database.

  11. Mass spectroscopy for protein identification MALDI-TOF spectrum MALDI-TOF Matrix assisted laser desorption/ionisation time-of-flight

  12. Generation of protein expression reference maps • Link protein information with DNA sequence information from the genome projects, • comprehensive 2D-gel databases constructed for different cell types • Listed at: • WORLD-2DPAGE: http//

  13. 2D-PAGE based differential expression proteomics • 2D-gel electrophoresis combined with mass spectrometry to get qualitative and quantitative protein behavioural data • Most frequently used method in proteome analysis from: Pandey and Mann, 2000

  14. Workflow of differential expression proteomics • Sample preparation • Isoelectrofocusing (1.dimension) • Equilibration incl. reduction, alkylation • SDS-PAGE (2. dimension) • Staining • Imaging • Spot detection and matching • Normalization and quantification • Analysis • Cutting of selected spots • Trypsin digestion in-gel • Identification with mass spectroscopy • Database comparison Steps to be practised during the workshop

  15. 2D-PAGE: critical points • Samples must be run at least in triplicate to rule out effects from gel-to-gel • variation → statistics • Standardized procedures needed to obtain a high reproducibility of 2D-gels • Sample preparation as • simple as possible • Isoelectrofocusing conditions • (patience) • Staining: fluorescent stains • for high sensitivity and high • linear range of detection Currently possible to run 12 gels in parallel

  16. Difference in-gel 2D-PAGE system (DIGE) • Proteins are labeled prior to running the first dimension with up to three different fluorescent cyanide dyes (Unlu et al.1997) • Allows use of an internal standard in each gel which reduces gel-to-gel variation, reduces the number of gels to be run • Adds 500 Da to the protein labelled • Additional postelectrophoretic staining needed from Kolkman et al. 2005

  17. Limitations and challenges of gel-based approaches • Dynamic range detectable on 2D-gels: 104, protein expression levels of a cell can vary between 105 (yeast) and even 1010(humans) enrichment or prefractionation strategies needed to reach less abundant proteins • Resolution of 2D-gels has its limits use narrow pH range gels and combine • Protein extraction and solubility during IEF can be a problem for poorly water-soluble proteins e.g. membrane proteins or nuclear proteins • Challenges for further development in gel-based proteomics: improve sample preparation to be able to analyze extreme proteins (extremely basic or acidic, extremely small or big, extremely hydrophobic), sensitivity, dynamic range, automation

  18. Copies/cell Proteome coverage - 106 - 20% - 105 - 40% - 104 - 60% - 103 - 102 - 80% - 101 - 100% 2D-gel based proteomics: the state-of-the-art versus the challenge

  19. Other proteomic approaches • Liquid chromatography coupled to mass spectrometry - Shotgun multidimensional protein identification technology MudPIT (Link et al. 1999) - ICAT: isotope coded affinity tags (Gygi et al. 1999), cysteine biased - iTRAQ (Ross et al. 2004): amine specificlabelling of peptides, quantification possible with tandem mass spectroscopy • Peptide and protein arrays (Lueking et al. 1999) • Yeast two-hybrid system (Fields and Song, 1989) • Phage display (Zozulya et al. 1999)

  20. ICAT for measuring differential protein expression ICAT consists of a biotin affinity group, a linker region that can incorporate heavy (deuterium) or light (hydrogen) atoms, and a thiol-reactive end group for linkage to cysteines. Proteins are labeled on cysteine residues with either the light or heavy form of the ICAT reagent. Protein samples are mixed and digested with a protease. Peptides labeled with the ICAT reagent can be purified using avidin chromatography. ICAT-labeled peptides can be analyzed by MS to quantitate the peak ratios and proteins can be identified by sequencing the peptides with MS/MS. from: Graves and Haystead, 2002

  21. 2D-PAGE in functionalproteomics Typical question: • Identify specific proteins in a cell that undergo changes in abundance, localization, or modification in response to a specific biological condition • Often combined with complementary techniques (protein biochemistry, molecular biology and cell physiology) If: - Monitoring quantitative changes in the biological process of interest - Quantitatively looking at protein modifications Then: 2D-gel based proteomics is the method of choice

  22. Zebrafish samples used for 2D-GE Experiment Phenotypic differences between untreated and treated zebrafish embryos • Treatment startet at high oblong stage of development • Samples taken from 70-90% epiboly stage

  23. Workflow of Differential Expression Proteomics • Sample preparation • Isoelectrofocusing (1.dimension) • Equilibration incl. reduction, alkylation • SDS-PAGE (2. dimension) • Staining • Imaging • Spot detection and matching • Normalization and quantification • Analysis • Cutting of selected spots • Trypsin digestion • Identification with mass spectroscopy • Database comparison Steps to be practised during the workshop

  24. 2D-Gelelectrophoresis Practical 3 different protein samples from: a) untreated embryos b) ethanol treated embryos c) selenium treated embryos Experiment 1: 7cm IPG strips 3-9 NL Passive rehydration/loading per sample 4 replicates, 12 strips run at the same time, has already been done  Start with equilibration and proceed to second dimension Experiment 2: 7cm IPG strips 3-9 NL Passive rehydration/loading per sample 2 replicates, 6 strips to be run 7cm IPG strips 7-10 Anodic cup loading to improve resolution of basic proteins per sample 2 replicates, 6 strips to be run Start with performing first dimension

  25. High performance analysis of 2D-gels 2D-gel analysis software practical -Introduction into the PDQuest software package -Demonstration of an comparative analysis of gels from two different sample types (wildtype, mutant) -Practising PDQuest analysis of the gels run during the workshop -Compare: a)control embryosvs. ethanol treated embryos b)control embryosvs. selenium treated embryos