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

1. 18/03/10
1
Plateforme de protéomique et d’analyse des
protéines par spectrométrie de masse
Université catholique de Louvain
Prof. Mark Rider
Prof. Pierre Morsomme
Head of the laboratories
Platform for proteomic and protein analysis by
mass spectrometry
2
Localized at the de Duve Institute and Institute of life Sciences at UCL
(http://www.uclouvain.be/en-proteomics.html)
Main equipments :
ThermoScientific LTQ XL linear ion trap mass spectrometer (CID + ETD)
Applied Biosystems 4800 MALDI-TOF-TOF
Capillary/nano 2D-LC systems
2D-gels (DIGE)
Molecular biology, protein purification (AKTA Explorer) …

2. 18/03/10
2
MASSPROT plaBorm in Woluwe
Historical overview
3
1997 MS set up in the HORM Unit by Mark Rider
Early work on protein and phosphorylation site identification
LCQ Classic
2002 Upgrade to a more sensitive MS
First LC-MS experiments
LCQ Deca XP Plus
LTQ XL + ETD
2005 Capillary/nano 2D-LC system
Proteomic gel-free work flow
2008 Linear ion trap with Electron Transfer Dissociation
Gas-phase radical ion fragmentation to preserve labile PTMs
top-down capabilities
MASSPROT plaBorm in Louvain‐la‐Neuve
Historical overview
4
1993 Protein sequencer
1996 2D-gels
2007 2D-LC-MALDI-TOF-TOF

3. 18/03/10
3
Platform for proteomic and protein analysis by
mass spectrometry
5
– Protein expression, purificaKon and characterizaKon
– Proteome analysis
– DifferenKal profiling and quanKficaKon of protein expression
– Post‐TranslaKonal ModificaKons discovery
Localized at the de Duve Institute and Institute of life Sciences at UCL
(http://www.uclouvain.be/en-proteomics.html)
Main equipments :
ThermoScientific LTQ XL linear ion trap mass spectrometer (CID + ETD)
Applied Biosystems 4800 MALDI-TOF-TOF
Applications:
Know-how of the platform
6
– Protein expression and purificaKon
– Expression in bacteria, yeast (S.cerevisiae, P.pastoris) and plants
– Soluble and membrane proteins purificaAon
Morsomme et al., J. Biol. Chem. 2002, 277, 29608

4. 18/03/10
4
Expression of a functional antibody in
Nicotiana tabacum plants and culture cells
7
– Expression in plants
– Transient / stable
– ConsAtuAve/inducible
Navarre et al., Transgenic Res. 2006, 15, 325
Expression of a functional antibody in
Nicotiana tabacum plants and culture cells
8
De Muynck et al., Transgenic Res. 2009, 18, 467

5. 18/03/10
5
Expression of a functional antibody in
Nicotiana tabacum plants and culture cells
9
NS0 SR1 BY2
HC
LC
Know-how of the platform
10
– Protein expression and purificaKon
– Expression in bacteria, yeast and plants
– Soluble and membrane proteins purificaAon
– Proteome analysis
– 2D‐gels + gel‐free (1D, 2D or 3D LC‐MS)
– Soluble and membrane proteins
– Non‐model species

6. 18/03/10
6
Proteome of non-model species: example of
Nicotiana tabacum BY2 cell culture
11
56 % of identification
Duby et al., Proteomics 2010, in press
795
1422
Know-how of the platform
12
– Protein expression and purificaKon
– Proteome analysis
– DifferenKal profiling and quanKficaKon of protein expression
– DIGE (2D‐gels)
– Isotopic labeling (iTraq, O18…)
– Label free quanAficaAon with spectral counts
 soluble proteins: E. Coli periplasm, glycosomes from
Trypanosomes
(Vertommen et al., Mol Biochem Parasitol. 2008,158:189)
(Vertommen et al., Proteomics. 2009, 9:2432)
 membrane proteins : E. Coli outer membrane
(Leverrier et al., Proteomics. 2010, 10:771)
 biomarkers of neurodegenerative diseases in human

7. 18/03/10
7
Know-how of the platform
13
– Protein expression and purificaKon
– Proteome analysis
– DifferenKal profiling and quanKficaKon of protein expression
– Post‐TranslaKonal ModificaKons discovery
 phosphorylation signal transduction
 glycation diabetes
 oxidation oxidative stress
 acetylation epigenetics
 ubiquitylation
 glutathionylation
 disulfide bridge
 glycosylation
 …
14
IdenKficaKon of Fructosamine‐3‐phosphate residues in human hemoglobin
 The aim of this work was to identify the fructosamine residues on
hemoglobin that are removed as a result of the action of FN3K in intact
erythrocytes

8. 18/03/10
8
15
Delpierrre G et al. J. Biol. Chem. 2004;279:27613
Three‐dimensional structure of human oxy‐hemoglobin showing the posiKon
of glycated residues
 First mass-spectrometric identification of
glycated residues in human hemoglobin
that are phosphorylated by FN3K
 Nine different fructosamine residues
phosphorylated by FN3K were identified
 The physiological importance of
deglycation is unknown at present.
Deglycation might prevent the further
conversion of fructosamines into advanced
glycation products
Platform for proteomic and protein analysis by
mass spectrometry
16
– Protein expression and purificaKon
– Expression in bacteria, yeast and plants
– Soluble and membrane proteins purificaAon
– Proteome analysis
– 2D‐gels + gel‐free (1D, 2D or 3D LC‐MS)
– Soluble and membrane proteins
– Non‐model species
– DifferenKal profiling and quanKficaKon of protein expression
– DIGE (2D‐gels)
– Isotopic labeling (iTraq, O18…)
– Label free quanAficaAon with spectral counts
– Post‐TranslaKonal ModificaKons discovery
– phosphorylaAon , glycaAon, acetylaAon, ubiquitylaAon,
glutathionylaAon, disulfide bridge, glycosylaAon …
http://www.uclouvain.be/en-proteomics.html

9. 18/03/10
9
17
Aknowledgements
Mark rider
Didier Vertommen
Pierre Morsomme
Marc Boutry
Hervé Degand
Anne-Marie Faber
http://www.uclouvain.be/en-proteomics.html