docSOMAscan™: A Quantitative Multiplex Proteomic
download 
Report Transcription
SOMAscan™: A Quantitative Multiplex Proteomic
SOMAscan™: A Quantitative Multiplex Proteomic Platform that Measures >1000 Analytes in Complex Matrices Tony Bartlett Director, European Commercial Operations & Collaborations, SomaLogic, Inc. 19th May 2014 Global Engage - Precision Medicine [email protected] THE TECHNOLOGY AN UNIQUE APPROACH TO PROTEOMICS & DISCOVERY OF DIAGNOSTIC BIOMARKERS 2 © 2014, SomaLogic, Inc. The technical challenge – proteomics trade-off? • To characterize biology, many proteins must be measured over a very wide range of abundances • Existing technologies require compromising either sensitivity or number of proteins 3 © 2014 SomaLogic, Inc. The usual “biomarker” paradigm Accurate representation of biological complexity Conventional Biomarker 4 © 2014, SomaLogic, Inc. The usual paradigm: often disappoints artistically Known surrogate endpoints used in routine clinical practice: CRP HBA1C FEV-1 HDL LDL Blood pressure Bone mineral density SVT PSA Recist Ham-D ADAS-Cog HIV viral load - the exception (causal?) Conventional Biomarker 5 © 2014, SomaLogic, Inc. A new paradigm: capture complexity by multiplexing True biological complexity Multi-dimensional biomarkers 6 © 2014, SomaLogic, Inc. The breakthrough technology: SOMAmers (slow off-rate modified aptamers) Modified side chains DNA backbone of SOMAmer 3’ Protein 7 1129 SOMAmers multiplexed = SOMAscan Intracellular 25 % Extracellular Domains 28 % Secreted 47 % Protease Hormones Structural Inhibitors 3% Proteins 5% 1% Growth Factors Receptors 13% 21% Proteases 17% Cytokines 20% Kinases 20% SOMAmer reagents cover a broad array of proteins associated with cellular processes and disease pathophysiology • Multiple compartments • Multiple pathways • 1,500-plex & 3,500-plex suggests no limit to capacity 8 SOMAscan: A protein to DNA transducer N n Protein Analytes 5000? Difficult to measure directly Formation of cognate complexes n SOMAmer Easy to measure 9 SOMAscan: A unique combination of attributes: High throughput & low volume • • • • • • • Measures 1129 proteins simultaneously Sample volume 65 µl Dynamic range ~8 logs Median lower limit of detection 40 fM (<1 pg/ml) Throughput of hundreds of samples/day High precision - <5% CV High specificity for individual, named proteins 10 © 2014, SomaLogic, Inc. SOMASCAN APPLICATIONS 1. MANAGING PRE-ANALYTIC VARIABILITY 11 © 2014, SomaLogic, Inc. Discriminating between real biology and pre-analytic variables Study in Healthy Volunteers – unbiased data analysis. Cellular Lysis Vector ~1000 Proteins Across All 80 Plasma Samples Differences due to sample handling 80 Plasma Samples In 4 Healthy Individuals (20 Samples Per Person) 1 Post Menopausal Female LH FSH 3 Males Sample time-tospin ranges from .5 hours (beige) to 20 hours (blue) Red = Intracellular neutrophil proteins Biological differences between 4 individuals Biology Vector 12 Avoiding false discoveries: Creating a criminal record for each sample • From the 1000 proteins measured we selected small sub-panels which were purely affected by only one type of pre-analytic effect • We created multidimensional vectors of the effects which are applied to each sample • We can use the vectors to include or exclude samples, and to include or exclude individual analytes Overriding contribution to signals in most studies – have to manage 13 SOMASCAN APPLICATIONS 2. DETECTING NORMAL VARIATIONS 14 © 2014, SomaLogic, Inc. Log protein fluorescence Response to a meal: decades of physiology exposed in a single study: Top proteins identified mathematically Fibroblast GF-19 Pancreatic Hormone Glucagon HAPLN1 Insulin-like GFBP1 Insulin Time in minutes after eating high fat meal 15 © 2013, SomaLogic, Inc. SOMASCAN APPLICATIONS 3. MECHANISTIC PHARMACOLOGY 16 © 2014, SomaLogic, Inc. Detecting a mechanistic response to pharmacology: iloprost (prostacyclin analog) in lung dysplasia Randomized placebo controlled study • 28 iloprost, 29 placebo • SOMAscan 1129 on paired plasma samples • Findings: – 14 proteins correlate to clinical response measured by bronchoscopy – 7/14 proteins are in the VEGF pathway – How iloprost is working or what drives dysplasia? Example single protein correlating with clinical response only seen if had ceased smoking 17 SOMASCAN APPLICATIONS 4. CARDIOVASCULAR DISEASE IN PRECLINICAL MODELS 19 © 2014, SomaLogic, Inc. Drug target discovery Keystone 2013 Richard T. Lee, M.D. Brigham and Women’s Hospital Harvard Medical School Harvard Stem Cell Institute Amy Wagers, Ph.D. Harvard Stem Cell Institute Harvard University Parabiosis is known to cure age-related heart failure – but how? • Cross-circulation is established 2-3 days after joining. • Blood chimerism reaches ~50% by 7-10 days. • Rapid exchange (~1%/min.) of cells and factors across the vascular junction. Wright, Wagers et al. Science 2001 SOMAscan found clear protein differences in old vs. young Lipidomics and metabolomics failed Age-related differences in GDF-11 were substantial (n=10/group) Cell. 2013 May 9;153(4):828-39. doi: 10.1016/j.cell.2013.04.015. Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy. 23 © 2014, SomaLogic, Inc. GDF-11 deficiency could cause age-related heart failure Intra-peritoneal injections of recombinant GDF-11 for 4 weeks reversed the phenotype in old mice similar to the effect of parabiosis Collaboration with Somalogic, Inc. © 2014, SomaLogic, Inc. 24 New England Journal editorial, Aug 8 2013 Cardiac aging and rejuvenation: A sense of humors? “These current findings add to a growing body of literature that suggests we might be entering a new era of “molecular humors.” 25 Independently, GDF-11 has previously been chosen as protein in a SomaLogic Dx panel Predicting cardiovascular event-free survival (death, MI, stroke, CHF) Framingham 11 Protein Discovery 11 Protein Validation 5-year event free survival curves by population quartile Successful discoveries by us, collaborators & clients Cancer: • Non-small cell lung • Pancreatic • Renal cell • Mesothelioma • Prostate Cardiovascular and Metabolic Disease: • Chronic kidney disease • Diabetic retinopathy • Heart failure • Liver fat and liver fibrosis (NASH) • Myocardial infarction • Stroke • Sudden death • Type 1 diabetes Inflammation and infection • Allergy • COPD • Inflammatory bowel disease • Pulmonary fibrosis • Rheumatoid arthritis • Infectious disease (TB, C-diff, cholera) Nutrition • Anti-inflammatory dietary supplement • Food and fat challenge Pharmacology • Doping with human growth hormone • Pharmacology of numerous drugs in cells, tissues and clinical samples • Prediction of drug response Other Diseases and Conditions: • Cystic fibrosis • Down syndrome • Duchenne muscular dystrophy • Pre-term birth Neuroscience: • Amyotrophic lateral sclerosis • Alzheimer's disease • Parkinson's disease Easy to find known and novel signals relating to pharmacology & disease! 27 © 2014, SomaLogic, Inc. 1129 What will you build? 28 © 2014, SomaLogic, Inc. HOW SOMASCAN WORKS 29 EQUILIBRATION WITH PLASMA/SE Streptavidin Coated Beads PreImmobilized SOMAmers PreImmobilized SOMAmers Non-specific SOMAmer:protein complex Cognate SOMAmer:protein complexes PROTEIN BIOTINYLATION NHS-Biotin BIOTINYLATED PROTEINS PHOTOCLEAVAGE 360 nm PHOTOCLEAVAGE 360 nm KINETIC CHALLENGE – Long half-life cognate complexes stay together Non-specific complexes dissociate quickly RELEASED COMPLEXES, FREE SOMAmers and FREE PROTEINS BIOTINYLATED PROTEIN CAPTURE Streptavidin Coated Beads CAPTURED BIOTINYLATED FREE PROTEINS & COMPLEXES – FREE SOMAmers WASHED AWAY ELUTE SOMAmers FROM COMPLEXES SOLUTION OF SOMAmers The number of each SOMAmer is a consistent fraction of the initial sample protein concentration SOMAscan: A protein to DNA transducer N n Protein Analytes 5000? Difficult to measure directly Formation of cognate complexes n SOMAmer Easy to measure 41 SOMAscan: Covers a broad range of sample matrices Routine/qualified Special/Research EDTA-Plasma Citrate-Plasma Serum CSF, Synovial fluid Tissue Homogenate Cell Lysates Conditioned Cell Media Plasma in non-human species Broncho-alveolar lavage Nasal lavage Tears Dried Blood Spots Urine Heparin plasma Whole blood Sputum 8 logs of protein concentration covered by 4.2 logs dynamic range per analyte Fine-tune [SOMAmer] to match [protein] in each of 3 sample dilutions Test a number of different samples (physiological concentration/genetic variation) 42
