LC MS2 - Proteomics/Metabolomics E. Waelkens BioSCENTer...

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Unformatted text preview: Proteomics/Metabolomics E. Waelkens BioSCENTer Doctoral School Technology Watch Course Clinical oriented research Biology oriented research Physiology/Pharmacology oriented research Food oriented research Chemistry oriented research Transcriptomics (micro-arrays) Proteomics Metabolomics Bioinformatics oriented research Genomics oriented research Proteomics = the study of the protein library- under certain well-defined conditions- in certain cells/tissues/ organisms Proteins Proteins Proteomics: What? Proteomics: What? Genomics =/=> Proteomics Proteins Proteins Genes Genes Proteomics Proteomics bB bB Genomics Genomics Proteins Proteins AA level Protein level •Proteolytic maturation •Methionine processing •Sugars, Ubiquitination •……. MODIFICATIONS ! Proteomics Proteomics Proteins Genes Metabolites Metabolomics Metabolomics 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 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 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 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 Flow Chart Flow Chart Separation Analysis Data processing Separation Separation MORE SEPARATION = MORE IDENTIFICATION b Combine separation steps: immuno-affinity based, subcellular fractionation, serum depletion kits, …. Typical Proteomics Typical Proteomics Separation techniques Separation techniques • 1D Gel (SDS-PAGE) • 2D GEL b 2D DIGE- GEL based separations- Liquid Chromatography (LC)- new separation techniques Separation Separation • 1D Gel (SDS-PAGE) • 2D GEL -> 2D DIGE GEL based separations Biringer & Amato, 2002 35 Separation Separation • 2D DIGE = labeling with fluorescing cyanine dyes: Cy2 (green) Cy3 (orange) Cy5 (red) GEL based separations 2D-DIGE , Separation Separation • 2D –Gel bB 2D LC • massive amount of data when linked to MS Liquid Chromatography (LC) New Separation techniques New Separation techniques • Special affinity based separations (special affinity matrixes: eg for serum samples/ membrane proteins/ phoshorylated peptides,...
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This note was uploaded on 02/16/2010 for the course CHEM 589 taught by Professor -1 during the Fall '02 term at Penn State.

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LC MS2 - Proteomics/Metabolomics E. Waelkens BioSCENTer...

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