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

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IBMM Proteomic Pla/orm
Université Libre de Bruxelles
Dr. Sabrina Bousbata*
Aimable Rengero Lema‡
*Head of the pla-orm
‡Technician of the pla-orm
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IBMM Proteomic Pla-orm ‐ 9 March 2010
Presenta?on Overview
• IBMM Proteomic Pla-orm Presenta?on
• Rou?ne Analysis: Protein Separa?on and
Iden?fica?on
• Research and Development

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IBMM Proteomic Pla-orm ‐ 9 March 2010
Strategies of IBMM Proteomic Pla-orm
http://nrpgm.sinica.edu.tw
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IBMM Proteomic Pla-orm ‐ 9 March 2010
IBMM 2‐DE Facility
IPGPhor Ettan Daltsix
ImageScanner Typhoon 9200
Flatbed 2nd dimension
1. 2-Dimensional gel Electrophoresis in different pH ranges
(4-7, 6-11, 3-10, narrow range)
2. Different type of staining in ng sensitivity
(coomassie, silver, sypro, proQ)
3. Image analysis and multivariante data analysis (SameSpot)

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IBMM Proteomic Pla-orm ‐ 9 March 2010
IBMM MS Facility
Waters Micromass MALDI-ToF-LR Waters Micromass CapLC Q-ToF ultima Global
1. Protein identification
Fingerprinting Sequencing
only simple samples
only with available genomes
simple but time consuming
complex samples
de novo sequencing
complicated but fast
2. Protein/peptide mass determination
with 0.1 % mass accuracy with 0.01 % mass accuracy
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IBMM Proteomic Pla-orm ‐ 9 March 2010
Classical Protein Separa?on and
Iden?fica?on

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IBMM Proteomic Pla-orm ‐ 9 March 2010
P1
P2
P3
P4
P5
P6
P7
P8
P9
Mw
IEBH
SDS‐PAGE Protein Separa?on and
Iden?fica?on
Smeesters P, Drèze PA, Bousbata S, Parikka KJ, Perez-Morga D, Timmery S, Toussaint A, Mahillon J and Van Melderen L (2009)
Genomic characterization of the circular prophage IEBH, a siphoviridae infecting Bacillus cereus. Submitted
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IBMM Proteomic Pla-orm ‐ 9 March 2010
4 pH 7
Yan JX et al., Proteomics 2002
DIGE gels
2096 spots > 1500 spots
4 pH 7
IBMM Coomassie gels
2‐DE Protein Separa?on and Iden?fica?on
All Proteins observed are identifiable

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IBMM Proteomic Pla-orm ‐ 9 March 2010
Differen?al 2D Gels
E.coli
LB medium
minimum glucose medium
1. csrA::kan vs. WT
2. ΔtldD+ΔtldE vs. WT
3. csrA::kan+ΔtldD+ΔtldE vs. WT
1. csrA::kan vs. WT
2. ΔtldD+ΔtldE vs. WT
22 spots
68 spots
91 spots
39 spots
50 spots
minimum glucose+casamino acids
1. csrA::kan vs. WT
2. ΔtldD+ΔtldE vs. WT
3. csrA::kan+ΔtldD+ΔtldE vs. WT
16 spots
48 spots
33 spots
All Differential Spots (300) have been identified
Interactomics by TAP‐Tagging
Galliot S, Brosson S, Bousbata S, Thiry M, Lafontaine D. (2010) Identification of a novel family of silver-stainable nucleolar protein
involved in nucleolar morphogenesis and cancer biology. In preparation

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IBMM Proteomic Pla-orm ‐ 9 March 2010
Research and Development
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Identified by MS and is immunoreactive
Immunoreactive
Identified by MS
80% sequence coverage
to discriminate isoforms with 96% identity
Immuno‐Based Spot Picking and
Protein Streaming Characteriza?on
Laugesen S, Bak-Jensen KS, Østergaard O, Roepstorff P, Svensson B (2007) Multiple forms of barley α-amylase generated during
germination identified by two-dimensional electrophoresis, immunoblotting, and mass spectrometry. FEBS J., 274: 2552-2565

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13 IBMM Proteomic Pla-orm ‐ 9 March 2010
DNGTY
LSEKGRFF
8180
4.61
21.3
C‐Terminal Processing Site Determina?on
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IBMM Proteomic Pla-orm ‐ 9 March 2010
Eluate
Total
FT
Soluble proteins Triton X‐114 phase
Total
FT
Eluate
116
97
55
36
31
66
Lysate
FT
Eluate
8/9e
Eluate
1/9e
49
38
116
97
55
36
31
66
Fig. 3: Anti‐Cruzipaïn Western Blot
Fig. 2: Enrichissment of T. cruzi Glycoproteins by
Lectin Affinity Chromatography
Tf TL Overlay
2 min
20 min
Fig. 1: Endocytic Pathway Stain of T. cruzi
a b c
Membrane Protein Enrichement and Lec?n
Affinity Chromatography

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Laugesen S, Messinese E, Hem S, Picheraux C, Rossignol M, Grat S, Ranjeva R, Rossignol M, and Bono J-J (2006)
Phosphoproteins analysis in plants: A proteomic approach. Phytochemistry 67:2208-2214
Phosphoprotein Enrichment and
Phosphoryla?on site Characteriza?on
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IBMM Proteomic Pla-orm ‐ 9 March 2010
Research in Progress
Gel‐Based Protein Quan?fica?on and ID:
1. Géné&que des Bactéries: Laurence Van Melderen, Johan Timmermans & Pierre
Smeesters
2. IMI: Michel Braun & Kathleen Weatherly
3. Delphi Gene&cs: Philippe Gabant & Céderic Szpirer
LC‐MS2 Quan?fica?on and ID:
1. Parasitologie: E?enne Pays, Luc Vanhamme, Didier Salmon, Sébas?en Brosson &
Pierrick Uzureau
2. Embryologie Moléculaire: Eric Bellefroid & Stephen Ghogomu
3. Service public fédéral Santé publique: Véronique Piehe
PTMs (Phosphoryla?on and Ubiqui?nyla?on):
1. Physiologie Moléculaire de la Cellule: Bruno André, Cédric Govaerts & Fabien
Debailleul
2. Biologie du Transport Membranaire: Anna Maria Marini & Mélanie Boeckstaens
3. Biologie Moléculaire du Gène: Véronique Kruys, Cyril Gueydan & Long Vo‐Ngoc