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Leigh Anderson PhD Plasma Proteome Institute

Protein Biomarkers for Personalized Medicine: Status and Prospects. Leigh Anderson PhD Plasma Proteome Institute. AAAS Personalized Medicine Colloquium June 1, 2009. The Plasma Proteome: Universal Diagnostic Potential?. Venezia. Credit: Space Imaging.

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Leigh Anderson PhD Plasma Proteome Institute

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  1. Protein Biomarkers for Personalized Medicine: Status and Prospects Leigh Anderson PhD Plasma Proteome Institute AAAS Personalized Medicine Colloquium June 1, 2009

  2. The Plasma Proteome:Universal Diagnostic Potential? Venezia Credit: Space Imaging • Plasma is the yellowish liquid part of blood left over when the cells are removed (serum is the liquid left after plasma has clotted) • Samples the imports and exports of nearly all cells in the body (like the canals of Venice) • Collected routinely for diagnosis • Large dilution volume: • ~2.5L plasma, • ~12L extracellular fluid • Variable protein lifetimes: • Some proteins disappear in minutes • Albumin lasts on average 21 days

  3. Protein Biomarkers in Plasma Have Shown Substantial Clinical Value

  4. Plasma Proteome Dynamic Range: >1010 in Concentration 3 logs 3 logs 4 logs * The Human Plasma Proteome: A Non-Redundant List Developed by Combination of Four Separate Sources, N. L. Anderson et al, Molec. Cell Proteomics, 3: 311-326 (2004).

  5. 1010 Really Is Wide Dynamic Range(Here on a linear scale) 10,000km 1000km 100km 10km 1km 10 9 8 100m 7 10m 6 1m 10cm 1cm 5 4 3 2 1 Slide courtesy Bruno Domon, ETH Zurich

  6. Observational History of Plasma Proteomics 10000 “3+-D” Number of Zones/Spots “2-D” Sequence Identified Unique Proteins 1175d 1000 607a 341b Number of Resolved Species 319c 100 60 49 “1-D” 40 10 1 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 • a. J. N. Adkins, et al, Mol Cell Proteomics1, 947-55. • b. R. S. Tirumalai, et al, Mol Cell Proteomics2, 1096-103. • c. R. Pieper, et al, Proteomics3, 422-32. • d. H_Plasma_NR-v2 Year The human plasma proteome: History, character, and diagnostic prospects. Anderson, N.L. and Anderson, N.G., Molecular and Cellular Proteomics, 1.11, 845-867 (2002)

  7. Discovery of New Protein Biomarkers Has Stalled: Only a Handful of New Protein Diagnostics Were Introduced in the Last Decade and at a Steadily Declining Rate

  8. Requirements for Biomarker Discovery • Two critical components: • Samples representing real clinical biology • Technology capable of finding real molecular differences • Questions have arisen regarding both components in the use of proteomics to discover cancer biomarkers

  9. Biased Samples Fatally Compromise Biomarker Studies Patient plasma samples Biomarker Candidates 1 2 Group A Proteomic Analysis Group B Cancer or Sample source? Failure Hospital 1 Ovarian Cancer Hospital 2 Control

  10. Instances of Sample Bias • Lancet ovarian cancer paper • Cancer and controls from different sources • Numerous instances in literature of bias due to • sample sources (e.g., cancer and control) • sex, age mismatches between groups • sample storage differences • Estimated to affect a majority of published protein biomarker studies • Erodes confidence in relevance of proteins as biomarkers

  11. Bias Is Now Understood and Can Be Eliminated • Gold standard approach: • Collect samples before diagnosis (cases and controls indistinguishable and hence treated same) • Select case and control samples from same intake population • All samples obtained using same collection, processing and storage protocol • Selected sample sets analyzed in randomized order on consistent platform • Changes related to clinical biology can be distinguished from other factors

  12. Unbiased Samples Allow Discovery of Real Biomarkers Patient plasma samples Biomarker Candidates 1 2 3 Group A Proteomic Analysis Group B Sample source Cancer Failure Hospital 1 Ovarian Cancer Hospital 2 Control

  13. Biomarker Candidates from the Literature: 1261 proteins related to Cancer A List of Candidate Cancer Biomarkers for Targeted Proteomics. Malu Polanski and N. Leigh Anderson, Biomarker Insights, 2: 1-48 (2006).

  14. Verification of Biomarker Candidates Is The Rate-Limiting Step in Generating New Diagnostics The Roles of Multiple Proteomics Platforms in a Pipeline for New Diagnostics, N. Leigh Anderson, Mol Cell Proteomics, 4: 1441-1444 (2005).

  15. Verification of Biomarker Candidates Is The Rate-Limiting Step in Generating New Diagnostics 1261 Cancer candidates: Cancer Informatics 2:1-48 (2006) 177 Cardiovascular candidates: J. Physiol., 563.1:23-60 (2005).

  16. Technology Alternatives for Directed Assaysfor Protein Biomarker Verification • Immunoassays (typical clinical test implementation) • Very sensitive • Expensive: IVD-quality assays cost $2-5 million • Specificity issues with less well-developed assays • Multiplexing limits in a single assay volume • Hybrid MS-based assays • Peptide MS for quantitation and identification • Insensitive to folding, complexation, etc. • Absolute analyte specificity • Multiplex 25-200 assays/analysis

  17. Mass Spectrometric (MRM) Quantitation of Peptides in Complex Sample Digests

  18. Multiple-Reaction Monitoring (MRM): Specific Mass Spectrometric Assay for Peptides • MRM • is a 2-stage MS assay capable of absolute analyte specificity with high precision (CV < 10%). • MRM measurement (QqQ-MS) is the most accurate quantitative MS mode • measures selected peptides in a sample digest as quantitative surrogates for the proteins from which they derive • assays for peptides can be designed directly from protein sequence • are implemented using triple-quadrupole mass spectrometers (QqQMS), very widely used for small molecular assays in plasma (drug metabolites, inborn errors, pesticides) • assays can be multiplexed (100+ assays per run)

  19. MS/MS Provides Absolute Assay Specificity:Peptide Identity Verified by MRM-triggered MS/MS MS/MS data from captured peptide Mascot interpretation of MS/MS data

  20. Many MRM Assays Can Be Carried Out at Once: 50 High-to-Medium Abundance Plasma Proteins Quantitative Mass Spectrometric MRM Assays for Major Plasma Proteins, Leigh Anderson and Christie Hunter, Mol Cell Proteomics, 5.4: 573-588 (2006).

  21. MRM Assays Are Reproducible Across Sites: CPTAC Verification Working Group Transition 1Transition 2Transition 3 X 4 reps

  22. MRM Sensitivity vsPlasma Dynamic RangeAntibody Capture of Analyte from a Large Mass of Digestvia SISCAPA

  23. SISCAPA™ : Increasing Target Peptide & Decreasing Ion Suppression

  24. Anti-Peptide Antibodies As Analyte-Specific Reagents VH VL 8-mer peptide bound to antibody groove N.K. Vyas et al (2003) PNAS 100:15023–15028. Anti-peptide antibody (APA) • Typically recognize 5-8 amino acid linear epitope in a combining site groove surrounded by complementarity-determining residues of Ig L and H chains • 6-8 amino acid sequences are typically unique in the human proteome • An anti-peptide antibody has the potential to select a single tryptic peptide from the human proteome • The peptide immunogen is easy to synthesize, and thus the antibody is easy to make (e.g., in rabbits)

  25. 10-Plex SISCAPA Capture(10 x 1μgpAb + 10μlplasma equiv digest) SISCAPA enriched P8 Hp (VGYVSGWGR) RBP (YWGVASFLQK) Albumin (LVNEVTEFAK) P7 CHAPS detergent P6 P2 P3 P1 P4 P5 Unfractionated plasma digest Target peptides are enriched (from near baseline) and other plasma peptides (Alb, Hp) are depleted

  26. Combining Patented Markers May Be Difficult • Obtaining multiple licenses from multiple sources increases difficulty geometrically • Royalty-stacking problem • How much of panel result is attributable to each separate marker (value of its IP) • There are few if any successful examples of multicomponent tests - ie no concrete experience • Possible solution via patent pools has never been successfully applied in medicine (though it is successful in electronics.

  27. A Large Library of MRM and SISCAPA/MRM Assays for Biomarker Development:the Human Proteome Detection and Quantitation Project (hPDQ)

  28. Human Proteome Detection andQuantitation Project: hPDQ • Objective: • To enable individual biological researchers to measure defined collections of human proteins in biological samples with 1 ng/ml sensitivity and absolute specificity, at throughput and cost levels that permit study of meaningfully large biological populations (~500-5,000 samples).

  29. Human Proteome Detection andQuantitation Project: hPDQ Four components: • A comprehensive database of proteotypic (protein-unique) peptides for each of the 21,500 human proteins. • At least two synthetic proteotypic peptides, labeled with stable isotope(s) and available in accurately quantitated aliquots. • Anti-peptide antibodies specific for the same two proteotypic peptides per target protein, capable of binding the peptides with dissociation constants < 1e-9. • Robust and affordable instrument platforms for quantitative analysis of small (amol to fmol) amounts of tryptic peptides and for sample preparation.

  30. Human Proteome Detection and Quantitation Project: hPDQ

  31. MRM & SISCAPA Assays Could Be Used in Clinical Laboratories -Eliminating One Translational Barrier to Adoption of New Biomarkers

  32. Initial Clinical SISCAPA Assay Hoofnagle et al, (2008) Clinical Chemistry 54:11 1796-1804

  33. Biomarker Tests Are Poorly Understood • Recent surveys indicate that the general public does not connect “in vitro diagnostic” with a blood test • Patients have no direct contact with IVD

  34. Protein Diagnostics Is a Very Small Part of Healthcare Protein-Based In Vitro Diagnostics Is Tiny Compared to Pharmaceuticals in Healthcare: ~1:100 Scale Difference

  35. Conclusions • Technology components of a rigorous biomarker development pipeline have been identified and are being implemented • New approaches to multiplex tests and longitudinal sampling may provide important further increases in diagnostic power • ….there is reason for optimism in biomarkers • However… • Major questions remain regarding the number and kind of protein biomarkers to be found • We do not understand why they finding them seems to be so difficult

  36. Acknowledgments Uinversity of Victoria, BC Terry Pearson , Angela Jackson, Matt Pope, Martin Soste, Lee Haines, Jamie Thomas, Department of Biochemistry and Microbiology, University of Victoria, B.C, Canada Derrick Smith, Darryl Hardy, Christophe Borchers, UVic-Genome B.C. Proteomics Centre Carr CPTAC Team Steve Carr, Eric Kuhn, Terri Addona, HazmikKeshishian, Sue Abbatiello, Broad Institute Mandy Paulovich, Jeff Whitaker, Lei Zhao, Fred Hutchinson Cancer Research Center MRM Assay Development Christie Hunter, Tina Settineri, Applied Biosystems, Foster City polySIS labeled peptide standards Lee Makowski, Frank Collart, Argonne National Laboratory Jerry Becker, Andrew Breite, Roche Protein Expression Group, Indianapolis • Antibody Development • Guoliang Yu, Xiuwen Liu, Epitomics • Kingfisher Magnetic Bead Processing • ThermoFisher • Triple Quadrupole Instrumentation • Applied Biosystems • Agilent Technologies • Animation • Arkitek Studios, Seattle • Grant Funding • National Cancer Institute • Biomarker Discovery Initiative (contract # 23XS144A) • Clinical Proteomic Technology Assessment for Cancer (grant U24-CA126476-01) • Canary Fund Seed Grant www.plasmaproteome.org

  37. Courtesy Steve Carr

  38. SISCAPA Publications • High sensitivity quantitation of peptides by mass spectrometry. Anderson, Norman L., United States Patent Application 20040072251. The basic SISCAPA patent application. • Stable Isotope Labeled Polypeptide Standards for Protein Quantitation.Anderson, Norman L., United States Patent Application 20060154318. Production of stable isotope labeled peptides as “polySIS” concatamer proteins. • Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). Anderson, N.L., Anderson, N.G., Haines, L.R., Hardie, D.B., Olafson. R.W., and Pearson, T.W. Journal of Proteome Research, 3: 235-44 (2004). Initial proof of concept results with SISCAPA using 4 peptides & Ab’s. • An effective and rapid method for functional characterization of immunoadsorbents using POROS® beads and flow cytometry. Anderson, N.L. , Haines, L.R. and Pearson, T.W. Journal of Proteome Research 3:228-34 (2004). Methods for characterizing SISCAPA immobilized Ab’s. • Candidate-Based Proteomics in the Search for Biomarkers of Cardiovascular Disease. Leigh Anderson, J. Physiol., 563.1:23-60 (2005). Explanation of the role of SISCAPA in biomarker validation and a list of 177 interesting candidate biomarkers in CVD. • The Roles of Multiple Proteomics Platforms in a Pipeline for New Diagnostics. N. Leigh Anderson, Mol Cell Proteomics, 4: 1441 - 1444 (2005). Role of SISCAPA assays in the Dx pipeline. • Quantitative Mass Spectrometric MRM Assays for Major Plasma Proteins. Leigh Anderson and Christie Hunter, Mol Cell Proteomics, 5: 573-588 (2006). Capabilities of QqQMS as the MS measurement platform for use in quantitating peptides/proteins in plasma digests: the SISCAPA detector. • Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers. J. R. Whiteaker, L. Zhao, H. Y. Zhang, L-C Feng, B. D. Piening, L. Anderson and A. G. Paulovich, Analytical Biochemistry 362:44–54 (2007). Implementation of SISCAPA on magnetic beads. • High-Abundance Polypeptides of the Human Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance. Glen L. Hortin, Denis Sviridov and N. Leigh Anderson, Clinical Chemistry 54: 1608-1616 (2008) • Protein Quantitation through Targeted Mass Spectrometry: The Way Out of Biomarker Purgatory? Steven A. Carr and Leigh Anderson, Clinical Chemistry 54:11, 1749–1752 (2008) • Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson,, J Immunol Methods, in press • SISCAPA Peptide Enrichment on Magnetic Beads Using an Inline Beadtrap Device. N. Leigh Anderson, Angela Jackson, Derek Smith, Darryl Hardie, Christoph Borchers, and Terry W. Pearson, submitted • A Human Proteome Detection and Quantitation Project: hPDQ. N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson, Christoph H. Borchers, Amanda G. Paulovich, Scott D. Patterson , Ruedi Aebersold and Steven A. Carr, in press

  39. Often One Marker Is Not Enough:Two or More Together Are Better Data replotted from Rifai, N. & Ridker, P. M. (2003). ClinChem 49, 666-669.

  40. Does Proteomics Technology Discover Real Molecular Differences? • Lancet paper (ovarian cancer) promoted a shortcut using immature technology (SELDI) that proved to be • not reproducible • not generally capable of identifying purported biomarker proteins • CPTAC is evaluating two streams of rigorous proteomic technology in terms of power and reproducibility: • “shotgun” proteomics for wide-angle discovery of candidates • “directed” proteomics for high-precision measurement of identified candidate biomarkers • Two basic questions: • Is proteomics capable of seeing cancer-related differences at all? • Are differences measured reproducibly in clinical samples?

  41. Initial CPTAC Answers - Shotgun • Shotgun (unbiased discovery) proteomics • does • detect a high proportion of real protein differences between samples • but does not • measure the same proteins in every run within a lab • measure all the same proteins in different labs • Hence the shotgun discovery approach is • appropriate for finding candidate biomarkers • not appropriate for testing candidates in rigorous clinical evaluation

  42. Initial CPTAC Answers – Directed • Directed proteomics • does • measure the same proteins in every run within a lab • measure all the same proteins in different labs • but does not • detect non-targeted (new) differences between samples • Hence the directed approach is • appropriate for testing candidates in rigorous clinical evaluation • not appropriate for finding new candidate biomarkers

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