Aplicació de la proteòmica a la cerca de Biomarcadors proteics Barcelona, 08 de Juny 2010

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1 Aplicació de la proteòmica a la cerca de Biomarcadors proteics Barcelona, 8 de Juny 21 Eliandre de Oliveira Plataforma de Proteòmica Parc Científic de Barcelona

2 Protein Chemistry Proteomics Hypothesis-free High-scale To identify as many components of the proteome as possible

3 The proteome is the set of proteins expressed from a determined genome. What we can do? Protein Mining: Identification of different proteins in a sample. Protein Expression Profiling: Comparison of protein expression under determined conditions. Protein Network Mapping: Approach to look at the interaction between proteins from different systems. Mapping of Protein Modifications: Identification of protein modifications and site mutation localization.

4 Applications Systems biology - understand cell-pathways, network, and complex interacting. Biological processes - characterize sub-proteomes such as protein complexes, cellular machines, organelles Biomarkers - discovery of disease (serological, urine, other biological fluids) - diagnostics, treat patients, monitor therapies Drug targets - evaluate toxicity & other biological or pharmaceutical parameters associated with drug treatment

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6 Protein Profiling Measure the expression of a set of proteins in two samples and compare them - Comparative proteomics 2-DE gel electrophoresis Difference gel electrophoresis (DIGE) LC-MS/MS using stable isotopic labeling (ICAT, itraq, SILAC..) ProteinChip (SELDI analysis) Antibody arrays

7 2-DE gels, DIGE Coomassie blue stained gels Silver stained - High resolving power - Absolute / relative quantity - Easily archived for further comparison - Detects some PTMs and alternatives splices - Low troughput - Poor detection of large, acidic, basic and membrane proteins - Only high abundance proteins Mix labeled extracts Internal standard

8 SELDI Surface Enhanced Laser Desorption Ionization Laser Chemical Surfaces (Hydrophobic) (Anionic) (Cationic) (Metal Ion) (Normal Phase) Biological Surfaces Ionized proteins are detected and their mass accurately determined by Time-of-Flight Mass Spectrometry (PS1 or PS2) (Antibody - Antigen) (Receptor - Ligand) (DNA - Protein) - High throughput - Small amounts of sample - More reproducible than 2DE, but lower resolving power - Applied for the analysis of crude samples - Process is not standardized

9 Antibody array Forward phase Reverse phase Sandwich assay Direct assay Detection with 2 nd Antibody Analyte Detection with Labeled Analyte Detection with Labeled Antibody Antibody immobilized on glass substrate Analytes immobilized on glass substrate - Not discovery based - Must have 1 or 2 specific high affinity antibodies - Very high throughput - Can be highly quantitative - relative and absolute - Can design reagents to detect PTMs, splice forms Modified from slide; FullMoonBiosystemsInc. (

10 Protein Identification by LC-MS/MS Digestion HPLC Protein mixture Peptides min MS LLTTIADAAK SAGGNYVVFGEAK EDDVEEAVQAADR Database Searching MS/MS m/z All peptide sequences Identification of many proteins

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12 Stable-isotope labeling Aebersold and Mann, Nature, 24

13 Isobaric tag reagent (itraq) Isobaric tags for relative and absolute quantification Allows us to compare the relative abundance of proteins from four different samples in a single mass spectrometry experiment Isobaric Tag (Total mass =145 Da) Peptide reactive group Reporter mass=114 to 117 Gives strong signature ion in MS/MS Balance mass 31 to 28 Balances the mass change of reporter to maintain a total mass of 145 Amine specific

14 Quantitative proteomics by using isobaric tag reagent-itraq PRG + P E P T I D E PRG+ P E P T I D E -PRG + P E P T I D E -PRG + P E P T I D E Mix MS NP E P T I D E 115 H 3 -NP E P T I D E H NP E P T I D E H NP E P T I D E H -Reporter-Balance-Peptide INTACT - 4 samples identical m/z MS/MS P E P T I D E - Peptide fragments EQUAL - Reporter ions DIFFERENT Mass (m/z) % Intensity A P 72.1 T 74.1 L q,h b b2 y2 T T T T Mass (m/z) y3 b4 y4 y5 b6 E E E E y6 D Mass (m/z) b7 D D D b8 y8 b9 y9 b1 y1 P P P P y11 E E E E P P P P

15 Approaches to Protein Biomarkers Single/Few Protein Assay(s) (Hypothesis) Multiplex Protein Assay(s) (Hypothesis/ Discovery) Candidate -Single -Panel Validation Value Added Proteomics (Discovery)

16 Detection Limits in Clinical Diagnostics for a 5 kdal protein analyte Biomarker Concentration pg/ml amol/ml Molecules/ml 5 mg/ml 5,,, 1,,, 6.2E+17 1 mg/ml 1,,, 2,, 1.2E+17 1 mg/ml 1,,, 2,, 1.2E+16 1 ug/ml 1,, 2,, 1.2E+15 1 ug/ml 1,, 2, 1.2E+14 1 ug/ml 1,, 2, 1.2E+13 1 ng/ml 1, 2, 1.2E+12 1 ng/ml 1, 2 1.2E+11 1 ng/ml 1, 2 1.2E+1 1 pg/ml E+9 1 pg/ml E+8 1 pg/ml E+7 Immunoassays QqQMS given 1- fold enrichment

17 Biofluid Proteomics Human plasma - Biomarkers of Sepsis (in collaboration with Dr. Ricard Ferrer and Dr. Antoni Artigas, Hospital de Sabadell) Human CSF - Biomarkers of ALS, Amyotrophic lateral sclerosis (in collaboration with Dra. Marta Vilaseca, Dr. Claudio Dilema and Dr. Joan Guinovart of the IRB, and Dr. Jacques Borg of Saint Étienne University Hospital)

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19 (Anderson, N. L., and Anderson, N. G. (22) The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, ).

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25 Am J Respir Crit Care Med Vol 178. pp 71 79, 28

26 Pipeline admission plasma sample Patient Selection discharge plasma sample Sample Preparation Protein Identification LC-MS/MS

27 LC-MS/MS Proteomics Clinical Plasma Samples Preparation & Digestion Peptides - Major plasma proteins Immunodepletion -Digestion -itraq labelling Ion intensity Liquid Chromatography Separation By Retention Time Retention Time Mass Spectrometry MS/MS Separation By Mass/Charge Measurement of Intensity Protein Identification Fragment Ion intensity Relative Abundanc e m /z Mass / Charge Ratio 14

28 - 4 sepsis patients - samples analyzed in two groups: (A1D1A2D2) + (A3D3A4D4) - Protein expression level is represented as a ratio: admission/discharge - ( abundance ratio)

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31 Pipeline Pre-treatment Plasma sample 1 Patient Selection Pos-treatment Plasma sample 2,3 Sample Preparation Protein Identification LC-MS/MS

32 LC-MS/MS Proteomics Clinical Plasma Samples Preparation & Digestion Peptides - Major plasma proteins Immunodepletion -Digestion -itraq labelling Ion intensity Liquid Chromatography Mass Spectrometry MS/MS Separation By Retention Time Separation By Mass/Charge Measurement of Intensity Protein Identification Fragment Ion intensity Retention Time Relative Abundanc e m /z Mass / Charge Ratio 14

33 Parc Científic de Barcelona Alex Campos Mª Antonia Odena Kostadin Evgeniev Atanasov Hospital de Sabadell Corporació Sanitària Parc Taulí: Dr. Antoni Artigas Dr. Ricard Ferrer Dra. Gemma Fuster Gemma Gomà Gisela Gili Thank you!