Stabilization Stabilizor Workflow Inactivation of Enzymes

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 POMC 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

6 5

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