QIAGEN Technologies for Proteomic studies

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1 Welcome QIAGEN Technologies for Proteomic studies QIAGEN Protein Seminar Dr Philippe JOANIN W W W. Q I A G E N. C O M

2 Why fractionate? Human proteome 100,000 different proteins No specific AntiBodies for each proteins 2D gel resolves proteins

3 Qproteome principles Making the proteome more manageable by: Fractionation = Sorting and re-sorting Depletion = Remove what you do not want Fractionation Depletion

4 Qproteome Kits Offer Toolbox To Study Dynamic Protein Modifications Sample Preparation Whole Proteomes Selective Depletion FFPE Tissue Kit Mammalian Protein Prep Kit Bacterial Protein Prep Kit Albumin/IgG Depletion Kit Murine Albumin Depletion Kit Sub-proteomes Physicochemical Properties Localization & Organelles Posttranslational modifications Soluble Protein Separation Kit Cell Compartment Kit Nuclear Subfractionation Kit Nuclear Protein Kit Mitochondria Isolation Kit Plasma Membrane Protein Kit PhosphoProtein Kit Total Glycoprotein Kit Mannose Glycoprotein Kit Sialic Glycoprotein Kit O-Glycan Glycoprotein Kit

5 Qproteome Kits Offer Toolbox To Study Dynamic Protein Modifications Sample Preparation Whole Proteomes Selective Depletion FFPE Tissue Kit Mammalian Protein Prep Kit Bacterial Protein Prep Kit Albumin/IgG Depletion Kit Murine Albumin Depletion Kit Sub-proteomes Physicochemical Properties Localization & Organelles Posttranslational modifications Soluble Protein Separation Kit Cell Compartment Kit Nuclear Subfractionation Kit Nuclear Protein Kit Mitochondria Isolation Kit Plasma Membrane Protein Kit PhosphoProtein Kit Total Glycoprotein Kit Mannose Glycoprotein Kit Sialic Glycoprotein Kit O-Glycan Glycoprotein Kit

6 Principle and Technology Qproteome FFPE Tissue Kit Remove paraffin and rehydrate FFPE tissue FFPE tissue Add Extraction buffer EXB followed by heat treatment Extracted Protein Transfer supernatant into a new tube after centrifugation Extracted Protein

7 Quantitative Extraction From Different Samples Sizes Liver Tissue (FFPE) Colon Tissue (FFPE) 250 Total pr otein recovered (µg) ß-actin Erk2 (p42) Sample size (mm 3 ) Six samples processed in parallel Data provided by Prof. Anja Bosserhoff, Institute of Pathology, University of Regensburg, Germany; 4 samples each size

8 Phosphorylation Detectable After Extraction Qproteome FFPE Tissue Kit Brain Tissue (Frozen & FFPE) Frozen FFPE Control

9 Qproteome Kits Offer Toolbox To Study Dynamic Protein Modifications Sample Preparation Whole Proteomes Selective Depletion FFPE Tissue Kit Mammalian Protein Prep Kit Bacterial Protein Prep Kit Albumin/IgG Depletion Kit Murine Albumin Depletion Kit Sub-proteomes Physicochemical Properties Localization & Organelles Posttranslational modifications Soluble Protein Separation Kit Cell Compartment Kit Nuclear Subfractionation Kit Nuclear Protein Kit Mitochondria Isolation Kit Plasma Membrane Protein Kit PhosphoProtein Kit Total Glycoprotein Kit Mannose Glycoprotein Kit Sialic Glycoprotein Kit O-Glycan Glycoprotein Kit

10 Extract The Phospho-Proteome PhosphoProtein Purification Kit Procedure Lyse Unphosphorylated Proteins in Flow-through Bind phosphorylated proteins Elute Phosphorylated proteins

11 Good Discrimitation Of Phosphorylated Proteins Western blotting of marker proteins Conclusions HSP-60 No retention of unphosphorylated proteins (A: HSP-60) Retention of phosphorylated proteins (B: MAPK) MAPK Protein-specific immunodetection of unphosphorylated HSP-60 protein and phosphorylated MAPK proteins. F: flow-through; E: eluate fractions.

12 Quantitative Retention Of Phosphorylated Proteins In vivo labelling of phosphoproteins 32 P labelled Jurkat Cells Conclusions Quantitative retention >95% of proteinous phosphor retained on column Data kindly provided by Gudrun Rehg and Sascha Dammeier, Altana, Konstanz, Germany

13 Thank You for Your Attention! Summary