The Clinical Potential of the Human Plasma Proteome

Leigh Anderson Ph.D. Founder & CEO, Plasma Proteome Institute Board Member, Dade Behring PPI The Plasma Proteome Institute Plasma is the largest, and deepest, version of the human proteome

• Largest = Most

• Deepest = Widest dynamic range

PPI Website Oct 2002 © Plasma Proteome Institute PPI Major Components of the Plasma Proteome • ~40,000 forms of proteins secreted to function in “Plasma Cellular Proteins plasma, most Proteins” (Leakage) – Assume 500 gene products x 2 splice variants x 20 glycoforms x 2 clip forms • ~500,000 forms of tissue proteins – Essentially all tissue proteins Immunoglobulins x splice and PTM variants • ~10,000,000 clonal forms of immunoglobulin

Total: the largest version of the human proteome

PPI Website Oct 2002 © Plasma Proteome Institute PPI A New Functional Classification of Proteins in Plasma

1. Secreted proteins that act in plasma (e.g., albumin, fibrinogen) 2. Immunoglobulins (Ig’s A,M,G,D,E) 3. Tissue leakage products (e.g., cardiac Mb) 4. “Distant” receptor ligands (e.g.,insulin) 5. “Local” receptor ligands (e.g., IL-8) 6. Aberrant secretions (e.g., PSA in cancer) 7. Temporary passengers (e.g., lysosomal enz.) 8. Foreign proteins (e.g., virus)

PPI Website Oct 2002 © Plasma Proteome Institute PPI Major Plasma Proteins 99% of plasma mass

Fibrinogen Transferrin IgA Total C4 Complement Complement Factor B Alpha-2-Macroglobulin Ceruloplasmin Prealbumin IgG Total IgM Total C9 Complement Alpha-1-Antitrypsin Factor H C3 Complement C1q Complement C8 Complement (a)

1% 10%

Alpha-1-acid

Apolipoprotein A-1

Albumin B 0 - 90% 90 - 99%

PPI Website Oct 2002 © Plasma Proteome Institute PPI Proteins Measured Clinically in Plasma Span > 10 Orders of Magnitude in Abundance 12

11

10

9 Dynamic range of single proteomic techniques 8

7 Total Cost: $10,695 6 per sample 5

4 Classical Plasma Proteins 3 Concentration in pg/mL Concentration Tissue Leakage 10 2 Interleukins & Cytokines

Normal Range Abundances Normal Range 1

Log Other 0 l l l r t I ) a s x 6 a 5 n 2 8 4 n n a F n n 1 T B ta D i i in in i ta - - - a - - r or h - n lin H 10 lin 12 se lin ti ta ta to es i ( S ein i ein ent m bin ide in ent ent - - bin ent ent ent ent ent ent or I 1 p Ig t gen A pha min t te te ct pha t bin* in - i l or B b psin ta o l or TPA nt erri in CEA iC3b i t kin kin l ote o m u lo m kin o otein kin kin ote m o o m ote t umin m m m m m a men c m ogen ogen f o Be olase r r r r r l To Tota ote c kin kin obu 1 be g ry obu g GC ept b s a n te 1 l l r le n le lobu le l - le le le le le le in i kin g Ferri P Pr P a P - it A To 1 a Ra eu eu o o eu eu eu n ag M r P

plasmin l

l g t l l l - a pha opon Alb op P F r ro - eu eu Inh r r r r r Fac m r b I opon on A l l n o Ig F og s pt c Ant c i IgG eu Ig lo P Comple r r r mp r i 1 mp mp r s mp mp mp mp mp l mp r C etop kin I T nt F My y TNF-Al te te sic te te te A r f F u M ing ent Tra sor Pr l e ent c T on Ga nt y a tive Pr - C e - -9 Pre f l te r te a ing P r ing G M cif Ha pop a In e h In r In In In te eu i cific E m b m u Bb e l P Pho Co Co Co nd -1 Co Co Co Co Co Co In In Tissue F T f *Hemo i r

olipoprotein L e M te nd Cer In r nd le a pe 9 q le 3 4 8 5 7 6 i - i p p pha ec id l te 5 1 olipoprotein S In C cid G B C C C C C C te l B A c - -2 - B pha p SC5 A C elin B C a -S mp In l mp C-reac Pr - In F A te n in no A i o x N c A t -1 My i us ta pha Complem Co l t o Co T e s Complem ur r a 2 A o R ta y 3 mb r pha C s h l Ne o P C o T A r P Thr

PPI Website Oct 2002 © Plasma Proteome Institute (Human male 50yr - from Specialty Laboratories Books) PPI Plasma “Proteomics” Began With 2-D Gels (c. 1976) 2-D Electrophoresis 300+ resolved spots 40 identified proteins

Anderson,L., Anderson,N. G. High resolution two-dimensional electrophoresis of human plasma proteins. (1977) PNAS 74, 5421-5 PPI Website Oct 2002 © Plasma Proteome Institute PPI Short History Most protein technologies have been applied to plasma rapidly

Year Event 1933 Von Mutzenbacher uses Svedberg’s analytical ultracentrifuge to resolve serum albumin and globulin fractions by molecular weight: demonstrates proteins are not heterogeneous colloids but rather specific structures 1937 Tiselius uses his electrophoresis to resolve serum proteins into α, β, and γ globulins, establishing a naming convention that still persists 1939 Tiselius and Kabat demonstrate that are components of the γ globulin fraction 1940’s Svensson and Longsworth further resolve serum globulins into α1, α2, β1, β2, γ1 and γ2 1950 Gofman finds float in ultracentrifuge and can be measured clinically 1950’s Paper electrophoresis of serum proteins is widely introduced into clinical chemistry 1958 Smithies and Poulik resolve 22 zones of serum proteins using starch gel electrophoresis 1960 Grabar and Burtin describe immunoelectrophoresis 1964 Ornstein and Davis introduce acrylamide ‘disc’ gel electrophoresis with resolution even higher than starch 1965 Laurell introduces crossed immunoelectrophoresis, resolving more than 40 different serum proteins 1966 Laurell introduces quantitative “rocket” electrophoresis 1977 Anderson and Anderson use 2-D electrophoresis to resolve hundreds of serum protein forms (40 identifications via immunoprecipitation) 1991 Published 2-D plasma protein database with 49 identified proteins and 727 spots 2002 Current SWISS-2D PAGE database with 60 identified proteins and est. 1500 spots 2002 Immunosubtraction/chromatography/2-DE with >250 identified proteins and est. 1000- 1500 spots (Pieper, et al, manuscript in preparation) PPI Website Oct 2002 © Plasma Proteome Institute PPI Growth in the Number of Protein Species Observed in Plasma Over Time

10000 “3+-D” Number of Zones/Spots Sequence Identified Unique Proteins “2-D” 1000 490+ 250* 100

“1-D” 49 60 40 10 Log10 Number of Resolved Species Log10 Number of 1 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010

Year

PPI Website Oct 2002 * R. Pieper et al, manuscript in preparation © Plasma Proteome Institute + J.N. Adkins, in press PPI 5'-Nucleotidase Collagen III c-terminal propeptide Growth Hormone Binding Protein Neuron-specific Enolase Acid labile subunit of IGFBP Collagen III n-terminal propeptide Haptoglobin alpha-1 Neutrophil-activating peptide 2 Acid phophatase, tartrate-resistant Collagen IV 7S n-terminal propeptide Haptoglobin alpha-2-chain Osteocalcin Acid phosphatase, prostatic Complement C1 Inhibitor Haptoglobin beta chain Haptoglobin beta chain, cleaved beta (from platelets) Complement C1q, A Pancreatic zymogen granule membrane Actin gamma (from platelets) Haptoglobin-related gene product Complement C1q, B Hemoglobin, alpha protein GP-2 Adenosine Deaminase Complement C1q, C Adiponectin Hemoglobin, beta Paraoxonase Parathyroid Hormone A Provisional Alanine aminotransferase (ALT) Complement C1r (Beta-1B-glycoprotein) Parathyroid Hormone-Related Protein Albumin Complement C1s Histidine-rich Alpha-2-glycoprotein PASP Aldolase (muscle type) Complement C2 ICAM-1, soluble Pepsinogen A Plasma Alkaline Phosphatase (bone) Complement C3A anaphylotoxin Ig Kappa light chain Plasma hyaluronan binding protein Alpha1,3-fucosyltransferase (FUT6) Complement C3B, alpha' Ig Lambda light chain Plasma kallikrein Complement C3B, beta IgA1 Plasma serine protease inhibitor Alpha-1-acid Glycoprotein IgA2 Proteome: Alpha-1-Antichymotrypsin Complement C4 anaphylotoxin IgD Plasminogen Alpha-1-Antitrypsin Complement C4, alpha IgE Plasminogen Alpha-1-B Glycoprotein Complement C4, beta IGFBP-3 Alpha-1-Microglobulin Complement C4, gamma IgG1 Pre-alpha trypsin inhibitor, H3 Alpha-2-Antiplasmin Complement C4-binding protein, alpha IgG2 Pregnancy-associated plasma protein-A 289 Proteins Alpha-2-HS Glycoprotein Complement C4-binding protein, beta IgG3 Pregnancy-associated plasma protein-A2 Alpha-2-Macroglobulin Complement C5A anaphylotoxin IgG4 Pregnancy-specific beta-1-glycoprotein 3 Alpha-fetoprotein Complement C5B, alpha' IgJ-chain Amylase (pancreatic) Prolactin Observed in Complement C5B, beta IgM Prolyl hydroxylase, alpha Angiostatin Inhibin (activin), beta A Angiotensin converting (ACE) Complement C6 Inhibin (activin), beta B Prolyl hydroxylase, beta Plasma Angiotensinogen Complement C7 Inhibin (activin), beta C Prostaglandin-H2 D-isomerase Antithrombin III (AT3) Complement C8, alpha Inhibin (activin), beta E Prostate Specific Antigen Apolipoprotein A-I Complement C8, beta Inhibin, alpha , H (Scientific Apolipoprotein A-II Complement C8, gamma Insulin C-Peptide Protein C, L Apolipoprotein A-IV Complement C9 Insulin, A chain Literature -100 Complement Factor B Insulin, B chain Protein Z Apolipoprotein B-48 Complement Factor B - Bb Fragment Insulin-like growth factor IA P-selectin, soluble -I Complement Factor D Insulin-like growth factor II Rantes Apolipoprotein C-II Inter-alpha trypsin inhibitor, H1 pre-2002) Complement Factor H Inter-alpha trypsin inhibitor, H2 Renin Apolipoprotein C-III Complement Factor I Retinol Binding Protein Apolipoprotein C-IV Inter-alpha trypsin inhibitor, H4 Connective Tissue Activating Peptide III Inter-alpha-trypsin inhibitorL S100 protein Corticotropin Releasing Hormone (CRH) Interferon Alpha Secretogranin V Apolipoprotein F C-reactive Protein Interferon Beta Apolipoprotein H Creatine Kinase, B Interferon Gamma Serum Amyloid P Creatine Kinase, M Interleukin-1 Beta Sex Hormone Binding Globulin Apolipoprotein J () Interleukin-10 Almost none Apolipoprotein(a) CRHBP Tetranectin Cystatin C Interleukin-12, alpha Aspartate aminotransferase (AST) Interleukin-12, beta were discovered Beta Thromboglobulin Elastase (neutrophil) Interleukin-1receptor antagonist Thyroid Stimulating Hormone Beta-2-microglobulin Eosinophil granule major basic protein Interleukin-2 Thyrotropin-releasing hormone CA 125 E-selectin, soluble Interleukin-4 Thyroxin Binding Globulin by proteomics CA 19-9 Ferritin, H Interleukin-5 Tissue Factor CA 72-4 Ferritin, L Interleukin-6 Tissue inhibitor of metalloproteinases-1 CA27.29/15-3 (MUC1 antigens) Fibrin fragment D-dimer Interleukin-8 Tissue inhibitor of metalloproteinases-2 Calreticulin Fibrinogen extended gamma chain IP-10, Small inducible cytokine B10 Tissue Plasminogen Activator Carboxypeptidase N , regulatory Fibrinogen, alpha Isocitrate Tissue Plasminogen Activator Inhibitor Carboxypeptidase N, catalytic Very little Fibrinogen, beta Kininogen TNF-Alpha Carcinoembryonic Antigen Ksp37 Cathepsin D Fibrinogen, gamma Laminin, alpha TNF-Binding Protein 1 published on CD5 antigen-like protein Fibronectin Laminin, beta TNF-Binding Protein 2 Ceruloplasmin Fibulin-1 Laminin, gamma Transcobalamin prediction of Cholinesterase Plasma Ficolin 1 LDH (heart) Chorionic Gonadotropin Beta (hCG) Ficolin 2 Lecithin- acyltransferase Transferrin secreted Chromogranin A Ficolin 3 Leucine-rich Alpha-2-glycoprotein Transferrin (asialo-, tau-, beta-2-) Chromogranin B (secretogranin I) Follicle stimulating hormone LHRH Transferrin Receptor (Soluble) Factor II (Prothrombin) G-6-PD Lipase Transthyretin proteins from Coagulation Factor IX Galactoglycoprotein (Leukosialin) Luteinizing hormone (LH), beta Triacylglycerol lipase (pancreatic) Coagulation Mannose-binding Protein Gamma-glutamyl transferase alpha Matrix metalloproteinase-2 Troponin I (cardiac) Coagulation Factor VII , H Gc-globulin Troponin I, (skeletal) Coagulation Factor VII, L M-CSF GCSF Melastatin Troponin T (cardiac) Coagulation Factor VIII Gelsolin Tryptase, beta-2 Coagulation MIP-1 alpha GHRH MIP-1 beta Tyrosine hydroxylase Coagulation Factor XI MSE55 Coagulation Factor XII Glutamate carboxypeptidase II Urokinase (High MW kidney type)A Glutathione Peroxidase Myelin Basic Protein Urokinase (High MW kidney type)B Coagulation Factor XIII A Myoglobin Coagulation Factor XIII B Glutathione S-transferase VCAM-1, soluble Glycoprotein hormones alpha chain N-Acetyl-B-D-Glucosaminidase, alpha Collagen I c-terminal propeptide N-Acetyl-B-D-Glucosaminidase, beta PPI Website Oct 2002 Collagen I c-terminal telopeptide (ICTP) GMCSF N-Acetylmuramyl-L-alanine amidase Von Willebrand Factor Collagen I n-terminal propeptide Growth Hormone Zn-Alpha-2-glycoprotein © Plasma Proteome InstituteCollagen I n-terminal telopeptide (NTx) PPI >70% of Proteins in Plasma Are Likely To Be In Complexes

Histogram of Masses of 262 Known Proteins in Plasma (masses of processed subunits computed from sequence) 30

25 Kidney 18% < 20kd Passed Retained 69% < 60kd 20

15

10

Number of Proteins Number of 5

0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 400 600 Mass Bin (kd) PPI Website Oct 2002 © Plasma Proteome Institute PPI Protein Diagnostics

• How does it compare to the discovery trend in proteomics? • What are the figures on protein tests approved by FDA (CLIA)?

PPI Website Oct 2002 © Plasma Proteome Institute PPI The Many Diagnostics Available Test For A Small Number of Protein Analytes

6,780 FDA-Approved Assays for 117 Different Protein Analytes in Plasma 400

350

300

250

200

150

100

50 Number of Approved Versions Number of Approved 0

Protein or Peptide Analyte

PPI Website Oct 2002 © Plasma Proteome Institute PPI Assays for Only 10 New Proteins in Plasma Have Been Approved by FDA Since 1993

50 2004 45 2002 40 35 2000 30 25 1998 20 1996 15 Using the Protein 10 Year of Introduction 1994 5

Number of Tests Approved 0 1992

9 ) II c c) - P -3 a) - a a or -3 ( ptor P 19 BN /15 e rdi rdi B 9 IGF 2 (Lp gen . a) , Ca IGF ti ( Rec T I (Ca 27 n -2 Recept An ei in in- n r errin n n ki gen rot sf ti p eu rl po Tropo Tropo Cance r An Li Tran te In

Cance

Based on the CLIA (Clinical Laboratory Improvement PPI Website Oct 2002 Amendments) Database maintained by the US Food © Plasma Proteome Institute and Drug Administration. PPI The Rate of Introduction of New FDA-Approved (CLIA) Diagnostic Protein Analytes Has Decreased to ~Zero

4

3

oteins in Plasma/Yr 2

1

0 Number of New Pr 1992 1994 1996 1998 2000 2002 2004 Year PPI Website Oct 2002 © Plasma Proteome Institute PPI Some Useful Protein Analytes Were Not Even “Identified” By the Standards of Proteomics: CA-125 Ovarian Cancer Marker

• Discovered in 1984 • Defined by monoclonal Ab(s) • 2000+ publications on clinical use • Identified with reference to protein sequence only in late 2001: – Tethered mainly-extracellular glycoprotein of 1,269,525 Daltons ∴ Proteomics ID standards not necessarily critical for applied use

PPI Website Oct 2002 © Plasma Proteome Institute PPI Expanding the Diagnostic Proteome

• A declining rate of introduction of new protein analytes contradicts the widespread expectation that genomics and proteomics are rapidly advancing non-genetic diagnostics • Suggests a major problem in translation of basic research into commercial diagnostics • PPI seeks to identify and overcome these barriers

PPI Website Oct 2002 © Plasma Proteome Institute PPI Some Barriers Impeding a Major Advance in Protein Diagnostics

Multivariate marker concept Not accepted Individual baseline concept Not accepted Cost per protein analyte 100x too high (for multi-protein markers)

PPI Website Oct 2002 © Plasma Proteome Institute PPI Multivariate Markers

• Available data indicates that multivariate (multi-protein) markers characterize disease states and drug effects better than single markers •Examples – Acute phase proteins in RA, in CV risk, in bacterial vs viral infections – CK-MB, Mb, TnI(T) in MI – Rodent tox studies of compound classes • Despite examples and theory, not enough weight of evidence to convince wide audience

PPI Website Oct 2002 © Plasma Proteome Institute PPI A Co-Varying Set of Protein Markers Yields a Disease Index More Sensitive and Robust Than a Single Protein Assay Relationship Between RA and Typhoid Vaccination Effects on Human Serum Protein Abundances

1.5

C3pro HpA2

1.0

Achymo 0.5

Cerulo

0.0

-0.5 Log10(RA/Control)

-1.0 Rheumatoid Arthritis Acute Phase -0.2 -0.1 0.0 0.1 0.2 Typhoid Vaccination Acute Phase log10(Typhoid, 48 hrs/baseline)

Analysis of Changes in Acute Phase Plasma Proteins in an Acute inflammatory Response and in Rheumatoid Arthritis using 2D-gel electrophoresis. NS Doherty, PPI Website Oct 2002 BH Littman, K Reilly, AC Swindell, Jane M Buss, NL Anderson, Electrophoresis. © Plasma Proteome Institute PPI Multivariate Protein Markers Resolve Drug Mechanisms As They Do Disease States Data from Quantitative 2-D Gel Studies in Mouse Liver With Test Panels Involving > 100 Proteins

F F G F G G Peroxisome FF G G Proliferator E G C E E E Treatments D E C D F A= Control se D spon n re C D B= LY163,443 ratio C A olife C D D A e pr C= LY171,883 A isom C A A erox A A P D= DEHP A A E= Clofibric acid AA A A AA F= WY14,643 L Y 1 G= Nafenopin 6 3 ,4 4 3

re s p o n s e B

B B B B B

Each symbol represents the liver protein pattern for 100+ proteins in the liver of an individual mouse Peroxisome Prolferators: 6 Compounds Compared Over 107 Selected Protein Spots The effects of peroxisome proliferators on protein abundances in mouse liver. PPI AndeWebsiterson, N. L.Oct, Esque 2002r-Blasco, R., Richardson, F., Foxworthy, P. and Eacho, P. © PlasmaToxicology Proteome and Applied Pha Institutermacology, 137, 75-89, 1996. PPI Plasma Markers: Monitoring Genetic Risk or Current Health Status? • Published data for different markers shows a wide distribution from almost total genetic control (unvarying levels) to none – Ratio of intra-individual to inter-individual CV’s (epidemiology studies) – % variation due to genetics (MZ twin studies) • Both methods show Lp(a) marker level is genetically determined: i.e., appropriate as a risk factor measure (assay needed one time) • Average proportion genetic is ~50% • Genetic component >20% suggests patient is best control for marker changes • Genetic component >80% suggests patient value will not change

PPI Website Oct 2002 © Plasma Proteome Institute PPI Intra-individual vs Inter-individual Coefficients of Variation for 16 Proteins in Plasma

100%

80%

60% CVi (Individual) 40% CVg (Group)

20%

0% Coefficient of Variation (CV) ) ) ) ) r n n n A n n 8 n a A 5 a i a i li i i - i t P 6 2 b ( b E p b p e P 2 h - il u in n o o o o b T 1 R lp L s m i l b C r l k r I ( e t t 1 a C a ( o t g lo i g u - ( - r r ll u C r 6 o o g to le n o h r y o L i in o t t p r k t n p o p g F e e c i r M a t u t k e o in a P p H n le o f u c i d I r r e p s le r L in e i r t s b n e e 2 - I v o t i r n n e t I i n c c e k o a u n m e le r r r r - o o C e h m CVi=CVg: Phenotype t - in x u e T e l S b lu CVi<

60% Attributed to Genetic 40%

20% Phenotype 0% I r I n t) y r- n Ia n or n E e a) to ct tein Ig ivit to g ( tide XI pti ige o ne ti n Proportion of Variation Proportion of Variation r ac e BP-3 fa actor II ac o f L F f o f tei pep ant d p I act an o ct c IG n copr II 1 r C- wth fi ra om X wth - p Fa o ci b owth c r o e e r gly o ipo ill h fic PAI L e gr e g ct e gr lik W ik - n -lik Fa -l n o e-ric n li v lin n -speci li u u u s s s In Prostate-sp In roup In * Histidi * Gc (g PPI Website Oct 2002 (Published values from many sources © Plasma Proteome Institute *= two discordant studies) PPI Individual Variation in CRP Over Time: Stability of Non-Disease Levels Allows Finer Characterization of Disease

1.8 CRP Levels Over Time in Three Individuals 1.6 A “cold” 1.4 1.2 ug/mL) 1 0.8 0.6

vel (Log10 0.4 0.2

CRP Le 0 -0.2 -0.4 12345678 Time Point (3 Week Intervals) Macy, E. M., Hayes, T. E. and Tracy, R. P., Variability in the measurement of C-reactive protein in healthy subjects implications for reference intervals and epidemiological applications. Clin. Chem. 43, 52-8 (1997) PPI Website Oct 2002 © Plasma Proteome Institute PPI Conclusions

• Diagnostic value of plasma proteome measurements substantially enhanced by – Use of more protein markers – Use of multiprotein panels – Use of patient as self-control • Current data is convincing but sparse • A series of demonstration studies is needed affect change in consensus view • Regulatory issues need debate • Integration of the above is timely

PPI Website Oct 2002 © Plasma Proteome Institute PPI Plasma Proteome Institute

• Purpose Expand the range of protein analytes and indications through application of rapid proteomics quantitation systems to sets of well-characterized clinical samples •Aims Promote multivariate protein tests Promote repeatedly sampling of individuals for detection of trends Advance technologies for routine plasma proteome measurement

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