Denator Novel Tissue Sample Preparation Technology for MS Analysis denator.com WATERS 2nd NORDIC MS SYMPOSIUM Tuesday Nov 8 2011 Båstad Katarina Alenäs Denator 2004 University spin-off 2006 Head office at Biotech Center Gothenburg, Sweden Research lab in Uppsala Science Park, Sweden 2008 Stabilizor T1 – Launch 2009-to date Publications: Svensson et al. “Heat stabilization of the tissue proteome” Journal of Proteome Research, 2009 Robinson et al. “Assessing the use of thermal treatment to preserve the intact proteomes of post-mortem heart and brain tissue” Proteomics, 2009 Scholz et al. “Impact of temperature dependent sampling procedures in proteomics and peptidomics” Molecular and Cellular Proteomics, 2010 Goodwin et al. “Stopping the clock on proteomic degradation by heat-treatment at the point of tissue excision” Proteomics, 2010 Rountree et al “Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization” Proteome Science, 2010 Ahmed et al “Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization” Journal of Neuroscience Methods, 2011 Colgrave et al “Neuropeptide profiling of the bovine hypothalamus: Thermal stabilization is an effective tool in inhibiting post-mortem degradation” Colgrave et al, Proteomics, 2011 Protein research workflow Collection Sample prep Analysis Bioinformatics Stabilization Extraction 2D-gels Dissection Clean up MS Storage Enrichment Western blot Transport Trypsin cleavage ELISA Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, 2002 Schematic flowchart of the peptidomics experimental setup Brain dissection •Homogenization Peptide •Centrifugation extraction •10 kDa cut-off filtration •Concentration Nano LC •Desalting system •Separation •ESI-MS •mass information MS •relative intensity levels MS/MS •Collision induced dissociation •sequence information Data •Differential peptide display analysis •Identifications by database matching Microwave fixation system Focus MW energy Inactivate enzymes Prevent post-mortem changes Protein degradation - post sampling 0 min 10 min Svensson et al., Journal of Proteome Research 2009, 8(2), pp 974–981 Degradation Rapid increase of peptides -> the result of degradation “New” peptides were fragments from high abundant proteins min Sköld, et al., Proteomics 2007, 7(24), pp 4445 Peptide intensity post sampling Leu-Enk P OM C Leu-Enk POMC BetaBeta endorphin endor phin 140000 2500000 1200000 120000 2000000 1000000 100000 800000 1500000 80000 600000 60000 1000000 400000 40000 500000 200000 20000 0 0 0 0 1 3 10 01 12 33 104 01 12 33 104 0 1 3 10 min post-mortem min post-mortem min post-mortem The stability of neuropeptides post sampling Sköld, et al., Proteomics 2007, 7(24), pp 4445 The importance of sample handling Proteins, peptides and their modifications can change rapidly post sampling Standardization of sampling and rapid stabilization of molecules of interest is key for high quality protein studies Phosphorylated proteins are heavily involved in Signaling transduction and they change rapidly post sampling or during sample preparation if they are not stabilized Maintainor® Tissue Stabilizor® T1 Treatment/Storage Stabilization Stabilizor workflow Inactivation of enzymes Svensson et al., Journal of Proteome Research 2009, 8(2), pp 974–981 MD LCMS MALDI MS Imaging ELISA 2D gels RPPA Western blot Proteins and peptides - 2D-gels - MALDI MS Imaging -LCMS Proteomics – 2D-gels Collaboration with Professor Mike Dunn, UCD Conway, Ireland Sample handling is crucial Current workflow not satisfactory Mouse brain: Proteomics Stabilization results in: Locations of differentially expressed proteins 4 7 250 High molecular weight spots increase ) Low molecular weight spots kDa 3 decrease Identifications show less (x10 Wt. Mol. degradation in stabilized samples 16 ”Stabilization shows a favourable effect on the integrity of the mouse brain proteome affording increased abundance of several intact proteins and reduced fragmentation.” Robinson et al., Proteomics 2009, 19(9):4433-4444 Mouse brain: Peptidomics on MALDI-IMS Stabilized Untreated Mw 6723,5 Goodwin et al., Proteomics 2010, 10 (9):1751-61 Biomarkers of interest in xenografts FFPE Stabilized+FFPE Data courtesy of The Institute of Cancer Research, UK Peptidomics – Mass spectrometry Collaboration with professor Per Andrén at Uppsala University Mouse model of Parkinson Sample handling is crucial Nano-LC-ESI MS Somatostatin Proenkephalin A 198-209 Thymosin melanotropin α Leu-ENK Met-ENK-RSL Met-ENK-RF Thymosin Proenkephalin A 198-209 Somatostatin melanotropin α Met-ENK-RSL Leu-ENK Met-ENK-RF Untreated Stabilized Svensson et al., Journal of Proteome Research 2009, 8(2), pp 974–981 LTQ-Orbitrap MS Mouse brain peptidomics with LCMS x106 90000009 80000008 70000007 60000006 50000005 40000004 30000003 Intensity 20000002 10000001 Detected intensity Detected 0 Endorphin Nociceptin Cerebellin 4 Met-Enk-RSL CART 62-102 Stabilized Gastrin rel. Peptide Untreated In collaboration with Dr. Capdevielle, Sanofi-Aventis, France Phosphorylations - Western Blot - Mass spectrometry Phosphorylation levels in clinical samples – up or down regulation can distort the analysis Espina et al., Molecular and Cellular Proteomics 2008, 7, pp 1998-2018 Western blot Phosphorylated proteins D MW 1 3 10 4000,0 3500,0 3000,0 2500,0 Denator Denator+2h RT 2000,0 SF + 10 min 1500,0 1000,0 500,0 Comparison between focused 0,0 MW and Denator in combination with a time course study. Level of phosphorylated protein Level of phosphorylated Brain-GSK Brain-CREB Brain-MAPK Svensson et al., Journal of Proteome Research 2009, 8(2), pp 974–981 Nano-LC-ESI MS Phosphorylated peptides Phosphorylated peptides 8 7 6 5 4 3 2 Relative abundance Relative log^2 1 0 Untreated Fresh treated Frozen treated Secretogranin-1 derived phosphorylated peptides In collaboration with Karolinska Institutet Reversed Phase Protein Arrays Stabilization preserves PTM’s for 24hrs Stabilizor T1 Instrument Heat system • Optimized conductive heat • Homogenous and rapid Vacuum system • Fully automatic • Optimizes the heat transfer Treatment time assessment • Ensures total inactivation • Ensures reproducibility Tracking system Info Size: 465 x 306 x 143 mm (l x w x h) Weight: 6,7 Kg Requirements: 110/220 volt Maintainor Tissue • For treatment and storage • Facilitates rapid and effective sample collection • Ensures effective heat transfer during treatment • Provides optimal conditions for transport and long-term storage Info Volume: 33 x 7 mm Stabilization Size: 85 x 54 x 2 mm Material : FEP (inert) Operating temp: -80 to 100 C CONFIDENTIAL Points of use Animal facility Stabilization of fresh tissue samples Operation room Stabilization of biopsies Stabilization of tissue from operations Lab Stabilization of frozen tissue samples Easy to use • Auto calibration • Pre-programmed methods • Portable Application support [email protected] +46 18 50 81 00 Some Stabilizor T1 publications “Assessing the use of thermal treatment to preserve the intact proteomes of post-mortem heart and brain tissue” Aisling A. Robinson, Jules A. Westbrook , Jane A. English , Mats Borén , Michael J. Dunn, Proteomics, 2009, 19 (9), pp 4433-4444 “Heat stabilization of the Tissue Proteome: A New Technology for Improved Proteomics” Marcus Svensson, Mats Borén, Karl Sköld, Maria Fälth, Benita Sjögren, Malin Andersson, Per Svenningsson, Per E. Andrén, Journal of Proteome Research, 2009, 8 (2), pp 974–981 “Impact of temperature dependent sampling procedures in proteomics and peptidomics-A characterization of the liver and pancreas post mortem degradome” Birger Scholz, Karl Sköld...Roman Zubarev, Peter James, Molecular and Cellular Proteomics, accepted Pre-print 27 Jan 2010 “Stopping the clock on proteomic degradation by heat-treatment at the point of tissue excision” Richard J.A. Goodwin, Alastair M. Lang…Andrew R. Pitt, Proteomics, 2010, 10 (9), pp 1751-61 “Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization” C Bart Rountree, Colleen V Kirk..Willard M Freeman, Proteome Sci. 2010; 8: 61 “Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization” Md. Mahiuddin Ahmed and Katheleen J. Gardiner, Journal of Neuroscience Methods, article in press January 2011 “Neuropeptide profiling of the bovine hypothalamus: Thermal stabilization is an effective tool in inhibiting post-mortem degradation” Colgrave et al, Proteomics 2011, 11, 1–13 “Benefits of heat-treatment to the protease packed neutrophil for proteome analysis: Halting protein degradation” Kennedy et al, Proteomics 2011, 11 (10) published online Apr 8 "Thermal Stabilization of Tissues and the Preservation of Protein Phosphorylation States for Two-Dimensional Gel Electrophoresis” Smejkal et al, Electrophoresis 2011 published on line April 2011 Some Stabilizor T1 users worldwide CSRIO, Livestock Industries, Australia Universidade de Vigo, Spain Capital Medical University Beijing, China Uppsala University, Sweden Copenhagen University, Denmark Karolinska Institutet, Sweden NovoNordisk, Denmark Leiden Medical University Center, Netherlands Helsinki University, Finland Delft University of Technology, Netherlands Turku Biotechnology Center, Finland St.George’s Biomics Center London, UK Max-Planck-Institute, Germany MerckSharpDome, USA University College Dublin, Ireland Penn State University, USA Tokohuso University Hospital, Japan University of Colorado, USA University of Fribourg, Switzerland University of Pennsylvania, USA Summary Protein degradation is rapid and may cause problems downstream Standardization within sample collection is important Rapid and homogenous heat inactivation is an efficient technique to keep the sample as close to its in-vivo state as possible Acknowledgements Professor Michael J. Dunn, Conway Institute of Biomolecular & Biomedical Research, Ireland Professor Per Andrén, BMMS, Uppsala University, Sweden Professor Andrew Pitt, Dept. of Integrative and Systems Biology, Glasgow University, UK Dr Per Svenninsson, Dept. of Physiology and Pharmacology, Karolinska Institutet, Sweden Dr Joel Capdevielle, Sanofi-Aventis, France denator.com www.denator.com.