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O B E K O N Consortium. Application of proteomics methods in the identification of biomarkers, suitable for studying obesity and obesity related diseases. OBECON Consortium. Identifying clinically relevant biomarkers. Biomarker applications. Drug Development. Disease management.
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O B E K O N Consortium Application of proteomics methods in the identification of biomarkers, suitable for studying obesity and obesity related diseases OBECON Consortium CECON II. Budapest
Identifying clinically relevant biomarkers Biomarker applications Drug Development Disease management Stratification markers Efficacy biomarkers Differentiation markers Toxicity biomarkers Screening markers Prognostic markers CECON II. Budapest
Multivariate panels versus singlebiomarkers • New tests are likely to increasingly focuson multivariate panels • Multivariate patterns appear more robustthan single analyte tests (less sensitive toindividual difference and assay noise) • The rational behind is well known in theclinical field (physicians generally integrate multipletest results to arrive to the diagnosis) • Identification of the proteins/analytes inthe panel is desirable but not critical • Regulations are currently being put inplace (MammaPrint™, FDA regulationson In Vitro Diagnostic Multivariate IndexAssays) CECON II. Budapest
Genome vs. proteome • Human Genome = 20 - 30,000 genes • Human Proteome = 300,000 to 1,200,000protein variants • Genome – static; proteome - dinamic; • „… there is only a 0.4 correlationbetween global mRNA and proteinexpression..” • Metabolic Profiling,ed.G.G.Harrigan, p72, 2004 • PTM: • Phosphorylation, Acetylation, Methylation, Hydroxy amino acids, Acylation, Myristic acid, Palmitic acid, Prenylation, Farnesol, Geranylgeranol, Nitrosylation, Oxidation, Other oxidation: loss of SH, Dityrosine formation, Isoaspartate, Glycation variable, Glycoxidation variable, Lipid peroxide adduction variable • Other variations: • Presenve of ligand, oligomeric state, cellular localization, proteolytic form; • Protein function determines phenotype: • “Catalytic” proteins, including enzymes,transporters, chaperons, etc. • “Structural” proteins CECON II. Budapest
Proteins are probably the best biomarkers • Proteins are at the site of action (they do the job, catalytic proteins); • They make up lots of structural elements of the organisms; • Respond to environmental and pathological changes with modifications; • The plasma carries the „message” from every tissue and cells;
Proteome - definition • If the genome is a list of the instruments in an orchestra, theproteome is the orchestra playing a symphony. • R.Simpson • Proteins and Proteomics:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2003, CECON II. Budapest
Challanges in proteomics 1. Protein function and activity; 2. What are the differences between the examination of proteins and nucleic acids? Speed, order of magnitude, sensitivity, reliability; 3. Maybe minor proteins are themost important but their examination is difficult with present day techniques; 4. There is no protein PCR; CECON II. Budapest
Research strategies for proteome-derived biomarkers • MS-based techniques • reproducibility, sensitivity, dinamic range and throughput; transformation to clinical test; • Afinity reagents based approaches • pAB, msAB, mAB, rec(scFV-Fab), scaffold binders (ankyrin, armadillo, NA); • Antigen-analyte used for immunization and screening vs, analyte intended to be recognized (peptides, recombinant proteins or domains, PTM, folding, interactions); • BSI’s approach: • To create nascent mAB libraries;
Current, MS based workflow for translation of protein biomarker discovery into clinical practice mAB proteomics workflow
Proteomics strategy of OBECON Shotgun MS Protein ID Result verification on individual samples Plasma collection; Represent. sampling Depletion mAB-production Selection differentials LC-MS/MS Fraction generation Epitope ID Protein N-glyc CECON II. Budapest
Representative samples from OBECON plasma collectionNOND; OND; OD CECON II. Budapest
Balancing protein representation in plasma Multiple Lectin Affinity Column MLAC Normalization Ultra- Normalization Multiple Affinity Removal Column Hu7 Hu14
Balancing protein representation in plasma 1----Total plasma 2----Hu7 depleted 3---- Hu7 depleted, norm. 4--- Hu14 depleted 5--- Hu14 depleted, norm.
Proteomics strategy of OBECON Shotgun MS Protein ID Result verification on individual samples Plasma collection; Represent. sampling Depletion mAB-production Selection differentials LC-MS/MS Fraction generation Epitope ID Protein N-glyc CECON II. Budapest
Proteomics profiles of the OBE vs Control NP • abhydrolase domain-containing protein fam108b1 • alpha-2-antiplasmin • anti-oxldl immunoglobulin • complement factor properdin • gelsolin • giant protein p619 • heat repeat containing 2 • kiaa1662 • kininogen • lim and calponin homology domains-containing protein 1 • nsp5 • nucleoporin nup160 • tchp protein • titin • transmembrane protein 108 • tubulin • u3 small nucleolar rna-associated protein 15 • unnamed protein product 1 • unnamed protein product 4 • apolipoprotein a-ii • chorein 1d • collagen alpha 1(xvii) chain • dorsal neural-tube nuclear protein • fam91a1 protein • filip1l protein • g protein-coupled receptor 45 • homeobox protein cux-2 • hypothetical protein fp972 • inter-alpha-trypsin inhibitor heavy chain 1 • kiaa0357 • paternally expressed gene 3 isoform 2 • scavenger receptor class a, member 3 • transcription intermediary factor 1 • uncharacterized protein dkfzp667f0711 • unnamed protein product 3 • vacuolar protein sorting-associated protein 8 homolog • zonadhesin variant 3 • alpha-1b-glycoprotein • alpha-2-hs-glycoprotein • apolipoprotein a-i • apolipoprotein c-iii • apolipoprotein e • ceruloplasmin precursor (ferroxidase) • complement c1r • complement c3 • complement factor b • hemopexin • thrombin • transthyretin • vitamin d-binding protein • vitronectin OBE Control CECON II. Budapest Normalized plasma
Proteomics strategy of OBECON Shotgun MS Protein ID Result verification on individual samples Plasma collection; Represent. sampling Depletion mAB-production Selection differentials OGE-IEF, PGC -LC Fraction generation Epitope ID MS/MS Prot.,N-glyc CECON II. Budapest
Fractionation of plasma samples by OFFGEL isoelectrophoresis M ND D ND D ND D ND D OGE Fr# 5 5 6 6 7 7 8 8 CECON II. Budapest
Analysis of complex oligosaccharides using graphitized carbon LC/MS • Glycosilation – PTM; • Changes properties of proteins; • Congenital disorders • Affects cell-cell communication; • Divers and its effect pattern dependent; • Analysis challenging; CECON II. Budapest
Proteomics strategy of OBECON Shotgun MS Protein ID Result verification on individual samples Plasma collection; Represent. sampling Depletion mAB-production Selection differentials LC-MS/MS Fraction generation Epitope ID Protein N-glyc CECON II. Budapest
High medium Hybridoma technology
Screening Capture screening assay: minimal fold change detectionis1.5 (CV<10%) CECON II. Budapest
Antibodies from hybridoma supernatants recognizing OD... CECON II. Budapest
...OND... CECON II. Budapest
... and NOND plasma components. CECON II. Budapest
Antibodies can differentially recognize OD vs NOND... CECON II. Budapest
...or OND vs. NOND plasma components. CECON II. Budapest
Cloning selected hybridomas CECON II. Budapest
OD specific clones CECON II. Budapest
OND specific clones CECON II. Budapest
Antibody characterization • Cognate antigen determination • Necessary, because of the immunization strategy; • Protein ID • Immunorecognition conveyed 1-2D gel mediated isolation followed by MS identification; • Epitope ID CECON II. Budapest
Antigen ID: Direct Protein ID • Immune recognition • Simple immunoprecipitation • Multi-immunoaffinity Protein ID Platform: • Plasma preparation for chromatography • Two-step immunoaffinity chromatography • Isolation: • One or two dimensional electrophoresis • Image analysis/comparison • Identification • Antigen ID from gel slices or from eluted fractions via mass spectrometry Scientific meeting 2009
Plasma preparation mAb 1 mAb 2 mAb 3 mAb 4 Multi-immunoaffinity Column (MIA) Mixture of 8 mAb’s mAb 8 Individual column chromatography 1 3 mAb 8 2 Elution of specific bound antigens Elution of specific bound antigen Wash out of non-specific binding proteins Wash out of non-specific binding proteins Image comparison of 1 or 2 dimensional SDS PAGE of flow through and eluate In gel digestion, LC-MS analysis. Antigen ID: Direct Protein ID
Summary: proteomics method application • MS-based biomarker discovery • MS-shotgun expt: selectively appearing proteins in the different patient groups; • Analysis of plasma fractions for protein and glycosilation differences in human and in animal models; • Antibody library-based biomarker discovery • Six fusions; > 6000 hybridomas; • Screening median and high producers using OND, OD, and NOND plasmas • Cloning hybridomas differentiating among groups; • Characterization of purified antibodies (E-ID, P-ID); • Testing individual plasmas; CECON II. Budapest
OBEKON Consortium Acknowledgement Kurucz I.1, Kádas J.1, Tardieu N.2, Malderez C.2, Urbányi Z.3, Drahos L4., Vékey K.4, Flachner B.5, Cseh S.5, Takács L.1,2. 1BioSystems International (BSI) Ltd., Debrecen; 2BSI SAS, Evry, France; 3Gedeon Richter Ltd., Budapest; 4Hungarian Academy of Sciences, Chemical Research Center, Budapest; 5TargetEx Ltd., Dunakeszi; NKTH # NKFP-A1-2006-0048 CECON II. Budapest
