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LC MS2 - Proteomics/Metabolomics BioSCENTer Doctoral School...

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Proteomics/Metabolomics E. Waelkens BioSCENTer Doctoral School Technology Watch Course
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Clinicalorientedresearch Biologyorientedresearch Physiology/Pharmacologyoriented research Foodorientedresearch Chemistryorientedresearch Transcriptomics (micro-arrays) Proteomics Metabolomics Bioinformatics oriented research Genomicsorientedresearch
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Proteomics = the study of the protein library - under certain well-defined conditions - in certain cells/tissues/ organisms Proteins Proteins Proteomics: What? Proteomics: What?
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Genomics =/=> Proteomics Proteins Proteins Genes Genes Proteomics Proteomics barb2leftbarb2right barb2leftbarb2right barb2leftbarb2right Genomics Genomics
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Proteins Proteins AA level Protein level •Proteolytic maturation •Methionine processing •Sugars, Ubiquitination •……. MODIFICATIONS ! Proteomics Proteomics
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Proteins Genes Metabolites Metabolomics Metabolomics
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Challenges in Proteomics/ Challenges in Proteomics/ Metabolomics Metabolomics Labile modifications (eg phosphorylation) Dynamic changing (not static) Handling dependent (sample (pre)treatment !) Wide concentration range (no PCR equivalent) Localization dependent Better technology = more data Differential analysis and quantification
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Proteomics/ Proteomics/ Metabolomics Metabolomics : : MS Core technology MS Core technology Proteomics ( 2 “soft ionization” techniques: ESI and MALDI) Mass spectrometry Mass spectrometry Mass spectrometry Metabolomics: FTMS
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Proteomics: How? Proteomics: How? Various ways examples: (2D)- gel electrophoresis (old, old fashioned, but it still climbs up the mountains) immunological techniques [western blot , immunohistochemistry, fluorescence,...] chromatography: UV profiling fluorescent tagged proteins [full- length, (sub)cellular localization] Protein (Peptide) arrays Mass spectrometry
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Metabolomics Metabolomics : How? : How? Various ways examples: biochemical assays GC- MS LC- MS CE- MS LC- NMR LC- EC (electrochemical array) FT-ICR- Mass spectrometry
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Flow Chart Flow Chart Separation Analysis Data processing
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Separation Separation MORE SEPARATION = MORE IDENTIFICATION barb2right Combine separation steps: immuno-affinity based, subcellular fractionation, serum depletion kits, ….
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Typical Proteomics Typical Proteomics Separation techniques Separation techniques 1D Gel (SDS-PAGE) 2D GEL barb2right 2D DIGE - GEL based separations - Liquid Chromatography (LC) - new separation techniques
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Separation Separation 1D Gel (SDS-PAGE) 2D GEL -> 2D DIGE GEL based separations Biringer & Amato, 2002 35 35
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Separation Separation 2D DIGE = labeling with fluorescing cyanine dyes: Cy2 (green) Cy3 (orange) Cy5 (red) GEL based separations 2D-DIGE ,
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Separation Separation • 2D –Gel barb2leftbarb2right 2D LC • massive amount of data when linked to MS Liquid Chromatography (LC)
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New Separation techniques New Separation techniques Special affinity based separations (special affinity matrixes: eg for serum samples/ membrane proteins/ phoshorylated peptides, antibodies based separations, dendrimer capture). Popular
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