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Plateforme protéomique GIGA - ULg - Lab'InSight proteomics
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Transcript of Plateforme protéomique GIGA - ULg - Lab'InSight proteomics
18/03/10
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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
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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
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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
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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
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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
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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
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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
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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
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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))
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R/D and Custom services
NanoparBcles synthesis and funcBonalisaBon
19 University of Liège ‐ 9 March 2010
Biosensors nanoRods
100nm
+ 100µm
Spheriods as in vivo model