Plateforme protéomique GIGA - ULg - Lab'InSight proteomics

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description of the laboratory skills (09/03/2010)

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Plateforme protéomique GIGA - ULg - Lab'InSight proteomics

  1. 1. 18/03/10 1 Plateforme protéomique GIGA  Prof. De Pauw Edwin  Head of the mass spectrometry laboratory  Members  2 University of Liège ‐ 9 March 2010  LSM – CART Lab. of Mass spectrometry Centre of Analytical Research and Technology E. De Pauw CNCM Center for Cellular And Molecular Neurobiology P. Leprince MEDICAL CHEMISTRY MP Merville M. Fillet
  2. 2. 18/03/10 2 Staff  Postdocs Marie-Alice Meuwis (Biomedical) Gabriel Mazzucchelli (Qauntification) Nicolas Smargiasso (Glyco) Delphine Debois (imaging) Quinton Loic (Venomics) Andrei Turtoi (GIGA Cancer) Research Scientist Anne-Cécile Massart Technicians Sabrina Llabrozzo Lisette Trzpiot Laurianne Wilmotte Irene Defaweux Gregory Gridelet  3 University of Liège ‐ 9 March 2010  Philosophy  • Research driven high quality service • Service activities within a quality managment environment • High end instrumentation dedicated expertise • Flexible collaborative schemes, from "standard" service to custom development and consulting 4 University of Liège ‐ 9 March 2010 
  3. 3. 18/03/10 3 Standard services  Gel separaBon methods  1D (mini, midi, native gel) 2D (mini, midi and 18x24cm gel format) 2D DIGE (18x24cm gel format)  5 University of Liège ‐ 9 March 2010  Spot Picker (GE)Ettan Dalt electrophosesis system Typhoon scanner TM9400 (GE) Standard services  Chromatographic methods for protein separation (LC-MALDI) 6 University of Liège ‐ 9 March 2010 
  4. 4. 18/03/10 4 Standard services  Mass Spectrometric Identification of proteins Pure proteins (automated -96well) Proteins in complex mixture 7 University of Liège ‐ 9 March 2010  Automation : 96 well format Digester (Janus Perkin Elmer) MALDI-TOF-MS (Ultraflex II - Bruker) PMF- MSMS nanoLC-ESI-MSMS (Ultimate 3000 –Dionex , Esquire- Bruker) Standard services  Peptide or protein mass and heterogeneity determination MALDI-TOF-MS (Bruker) ESI-Q-TOF-MS (Waters) 8 University of Liège ‐ 9 March 2010  - PTM - S-S bridges - adducts - Peptide fragmentation Mass accuracy : 100ppm - protein isoforms - Protein complexes study - Peptide fragmentation - PMT and adducts Mass accuracy : 10ppm
  5. 5. 18/03/10 5 Standard services  Exact mass determination Elemental composition determination  9 University of Liège ‐ 9 March 2010  Mass accuracy < 1ppm Isotopic model comparison Elemental composition MS/MS experiment: CID, IRMPD, ECD, OPO UV-Vis Laser Structural information Standard services  Glycan analysis - Glycosylation of proteins - N- and O-Glycans repertoire determination (on cell extract or pure proteins)   10 University of Liège ‐ 9 March 2010  * MALDI-MS and MSMS on permethylated glycans * Developments towards relative quantitative analysis of permethylated glycans on nanoAcquityUPLC-SynaptTM HDMSTMG1 - Glycopeptides enrichment methods - Identification of Glycosylation sites on proteins
  6. 6. 18/03/10 6 Standard services  Edmann like sequencing Top-Down sequencing by ISD on MALDI- TOF-MS 11 University of Liège ‐ 9 March 2010  No need for trypsin digestion ! Standard services  Differential proteomics- clinical proteomics: peptides profiling with SELDI-TOF-MS (PCS 4000- BioRad) 12 University of Liège ‐ 9 March 2010  1. sample 2. Chip preparation 3. SELDI-T0F-MS spectra acquisition - High number of individual clinical samples - Peptides and proteins <25kDa - Low Quantities or V of biological fluids, tissues or cell lysats Satistical analysis with univariate and multivariate approaches (GIGA bioinformatic platform – R. Marée, P.Geurts) Need ID of peptides and small proteins ( purification and High resolution MS) Mass spectra or protein profiles m/z
  7. 7. 18/03/10 7 Standard services:   Quantitative proteomic profiling  13 University of Liège ‐ 9 March 2010  Ettan Dalt electrophosesis system Typhoon scanner TM9400 (GE) Statistical Analysis with: Decyder TM vs 7.0 (DEA, BVA) SameSpots Progenesis Automated Spot picking and ID of Protein by MS Standard services:   Quantitative proteomic  14 University of Liège ‐ 9 March 2010  in vivo labelling in vitro labelling SILAC ICPL 2D-nanoLC-ESI-MSMS (Ultimate 3000 - Esquire – Bruker) Analysis with Protein Scape (Bruker) ID and Relative quantitative analysis
  8. 8. 18/03/10 8 Standard services:   Label free quantitative proteomic  15 University of Liège ‐ 9 March 2010  R/D and Custom services  MALDI imaging  16 University of Liège ‐ 9 March 2010  Soon ! Small molecules imaging
  9. 9. 18/03/10 9 R/D and Custom services  Binding‐interacBons  17 University of Liège ‐ 9 March 2010  - Stœchiometry - Affinity - Study complex or protein « shape » (Ion Mobility) Telo 4: d(TTAGGG)4 NH4 + (5 µM DNA-10 µM drug) R/D and Custom services  18 University of Liège ‐ 9 March 2010  - Separation by IMS of proteins with same MW, charge but ≠ folding - Determination of cross section (Ω) of protein or complexes m/z Color = intensity The Drift time of protein depend on its m, z and its shape (cross section (Ω))
  10. 10. 18/03/10 10 R/D and Custom services  NanoparBcles synthesis and funcBonalisaBon  19 University of Liège ‐ 9 March 2010  BiosensorsnanoRods 100nm + 100µm Spheriods as in vivo model

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