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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