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

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. 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. 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. 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. 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. 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. 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. 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. 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. 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