Proteome Sciences Plc · LSE:PRM
Proteome Sciences — 7th Siena Meeting From Genome to Proteome: Back to the Future (September 2006)
27 slides from Proteome Sciences's conference presentation, Sep 5, 2006
Slide 1: The Impact of Mass Spectrometry on Alzheimer's Disease Research
Ps The Impact of Mass Spectrometry on Alzheimer’s Disease Research Ps Proteome Sciences 7th Siena Meeting From Genome to Proteome: Back to the Future 5th September 2006
Slide 2: Scope of this Talk
Ps Scope of this Talk • Introduction to Alzheimer’s Disease Pathology • The Need for Biomarkers • Application of ProteoSHOP™ Technologies to Biomarker Discovery • Tau Phosphorylation and New Therapeutic Targets • Towards Target Validation • Future Perspectives
Slide 3: Pathology of Alzheimer's Disease
Ps Pathology of Alzheimer’s Disease Alzheimer’s disease (AD) is characterised by a progressive neurological deficit with significant brain cell death Pathology features amyloid plaques together with neurofibrillary tangles A prolonged asymptomatic period precludes early intervention Mild Cognitive I
Slide 4: Ideal Biomarkers of AD
Ps Ideal Biomarkers of AD Detect a fundamental feature of neuropathology Validated in neuropathologically confirmed cases Sensitivity >80 % vs. controls Specificity >80 % vs. other dementias Reliable , reproducible & inexpensive Noninvasive & simple to perform Confirmed by at least two independent s
Slide 5: Current CSF Markers of AD
Ps Current CSF Markers of AD Amyloid β Aβ40 & Aβ42 levels decrease Tau total tau & phospho-tau levels increase Good sensitivity vs. controls Limited specificity vs. other dementias Early markers of AD ? Possible predictor of conversion from MCI/normal to AD Evidence of change with progression best f
Slide 6: ProteoSHOP Biomarker Discovery
Ps ProteoSHOP™ Biomarker Discovery Mission Statement To utilise multiple proteomics approaches to investigate protein changes in plasma samples from patients with Alzheimer’s disease compared to appropriate controls To further evaluate selected protein changes using immunological techniques Plasma c
Slide 7: Plasma Proteomics: A challenge
Ps Plasma Proteomics: A challenge
Slide 8: Choosing the best sample
Ps Choosing the best sample absorption and metabolisation blood in tissue intermediate tissue compartment pathologic events Csf renal synovial fluid) excretion proximity to disease Accessability concentration of analyte proteolytic processing Adapted from: Peptidomics in Drug Development, 1996, ECV
Slide 9: Proteins and Peptides
Ps Proteins and Peptides peptein = gr. to digest Adapted from: Peptidomics in Drug Development, 1996, ECV
Slide 10: Three study setups
Ps Three study setups 1. 2-DE 50 AD vs. 50 non-demented controls – whole plasma Single gels per sample, composite image, spot selection by MELANIE IV 30 AD vs. 30 non-demented controls - depleted plasma Single gels per sample, composite image, spot selection by Progenesis 2. Preliminary SELDI-TOF Pr
Slide 11: Biomarker Discovery: Quantitative Protein Sequence Tagging (qPST)
Ps Biomarker Discovery 3. Quantitative Protein Sequence Tagging (qPST) 30 AD vs. 30 non-demented controls - depleted plasma O * O 3 pools of 10 samples from AD and control groups * N * N CNBr pre-cleavage + reduction & alkylation * O Labelling with heavy and light qPST tags O Sample mixing, purifica
Slide 12: Discovery Phase Results (2DE)
Ps Discovery Phase Results (2DE) Outcome of 2DE Experiments Discovery cohort of 25 vs 25 217 common proteins 14 regulated between 1.5 - 2-fold at p<0.05 Validation cohort of 25 vs 25 4 markers replicated Protein identification by LC/MS/MS Merged cohort of 50 vs 50 Robust statistical analysis involvi
Slide 13: Discovery Phase (SELDI-TOF)
Ps 14000 14500 1 5 0 00 Discovery Phase (SELDI-TOF) 30 20 10 14646.2+H Cntrl Indv A Q10 854 14.6 kDa species 0 30 20 Cntrl Indv B Q10 854 10 2.29 fold increase in abundance in AD (p = 0.036) 14642.0+H 0 30 20 Cntrl Indv E Q10 854 10 14631.8+H 0 30 Material extracted from the chip surface → SDS-PAGE
Slide 14: The qPST Approach
Ps The qPST Approach control patients AD patients 30 plasma samples 30 plasma samples +/- Immuno- Depletion* * removal of haptoglobine, IgA, IgG, albumine, anti-trypsin, and serotransferrin 10 10 10 10 10 10 Control Case pool Control Case pool Control Case pool pool pool pool qPST qPST qPST Experime
Slide 15: Results of qPST Experiment
Ps Results of qPST Experiment 12 peptides from 8 proteins – Leading candidate biomarkers of AD
Slide 16: Summary
Ps Summary 1 Analysis of blood plasma samples: A) 2-D-PAGE in a cohort of 50:50 patients resulted in a selection of 14 protein changes with 4 confirmations. Proteins are in the µg-mg/mL range B) SELDI-analysis (4:4 patients) results in few changes which are all related to major abundant transport pr
Slide 17: Phase 2: Biomarker Evaluation
Ps Phase 2: Biomarker Evaluation Evaluation of Leading Candidates by Immunological-Based Techniques Western blot Complement Factor H (CFH) 121 AD patients CFH up-regulated in AD 205 non-demented controls Western blot validation 121 AD vs 148 controls 100 Motor Neuron disease (MND) patients Significa
Slide 18: Tau Phosphorylation and AD: The Concept
Ps Tau Phosphorylation and AD: The Concept • Tau is the main protein in neurofibrillary tangles • Phosphorylation of Tau is described for many sites of the molecule • Hyperphosphorylation of Tau is associated with increased disease severity • Kinases which potentially phosphorylate Tau at distinct s
Slide 19: Tau Phosphorylation and AD
Ps Tau Phosphorylation and AD Phase 1 Comprehensive discovery of mod sites (pSer, pThr and pTyr) Phase 2 Further Assessment using In vitro system Phase 3 New reagents and concepts (Peptides, Antibodies, Tandem Mass Tags) Phase 4 Design of animal model for validation of Biomarkers and Drug Targets De
Slide 20: Phosphorylation of PHF-Tau
Ps Phosphorylation of PHF-Tau Phase 1 Recap of data presented at Siena 2002 1 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT 51 PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG 101 TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK 151 IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SG
Slide 21: Tau Phosphorylation and AD
Ps Tau Phosphorylation and AD Hyperphosphorylated Tau is isolated from AD-brain tissue. After tryptic digestion, phosphorylated peptides are identified by LC/MS • In a first analysis, 19 phosphorylation sites were mapped to the Tau molecule. Subsequent improvements in MS-sensitivity including MRM le
Slide 22: Phosphorylation of Tyr394 in PHF-Tau Confirmed
Ps Phosphorylation of Tyr394 in PHF-Tau Confirmed Discriminating fragment ions present in MS/MS data One tryptic peptide carrying two phosphorylation sites which are selectively phosphorylated
Slide 23: Overview of Tau Kinases and phosphorylation sites
Ps Overview of Tau Kinases and phosphorylation sites Kinase PHF In vitro Literature Site of phosphorylation (pS and pT)
Slide 24: Summary
Ps Summary 2 • Several novel sites have been discovered which are selectively phosphorylated by Casein Kinase 1 (CK-1) • One site have been discovered at Tyr 394 which is selectively phosphorylated by the kinase c-Abl CK1 and c-Abl represent potential New Targets for AD therapy and have biomarker po
Slide 25: Steps Towards Validation of CK1 and cAbl
Ps Steps Towards Validation of CK1 and cAbl Based on these results, we further engage into 1. Production of Synthetic Peptides and Phosphopeptides 2. Making antibodies that recognise specific phosphorlyation sites 3. Generate transgenic animals to validate CK1 as relevant in AD-pathogenesis and a ta
Slide 26: Conclusions
Ps Conclusions The Impact of Mass Spectrometry on Alzheimer’s Disease Research Mass spectrometry shows increasing potential for AD-biomarker discovery AD-biomarker confirmation AD-biomarker validation Improvements in sensitivity and quantitation capabilities leads to highly improved discovery result
Slide 27: Acknowledgements
Ps Acknowledgements Proteome Sciences plc MRC Centre for Malcolm Ward Neurodegeneration Abdul Hye Research Helen Byers Simon Lovestone James Campbell Brian Anderton Annette Dalrymple Madhav Thambisetty Jules Westbrook Diane Hanger Vaksha Patel Hugh Reynolds Josef Schwarz Richie Williamson Stefan Kie