1. The World of Biomarkers Proteomics: Molecular Dissection Update on collaboration between Cris Dos Reemedios Jenny Van Eyk Mike Dunn Chip Petricoin

2. Goal To identify and characterize the extensive proteome changes that occur with end-stage heart failure compared to age matched controls.

3. Two Prong Experimental Strategy A) In-depth analysis of multiple subproteomes using multiple technologies for maximum coverage B) High through put validation of in-depth analysis of potential key pathways from previous whole myocardium analysis, literature and new proteomic data - CHIP (eg. myofilament) - reversed arrays (eg. AKT pathway) Dunn and Van Eyk Dos Reemedios and Van Eyk Dos Reemedios, Van Eyk and Petricoin Correlate to mRNA levels Correlate to functional data Validate potential candidate proteins (and pathways) Carry out further functional analysis of key pathways Develop new hypothesis

4. 1. In-depth analysis a)Optimize (and develop standardized operating procedures) for isolation for each subproteome. b)Determine the combination of technology platforms that provides maximum proteome coverage c)Optimize technologies to minimize technical error. 2. Focused and validation analysis a)Determine proteins of interest and test Ab for specificity b)Optimize antibody and cellular extract (or subproteomes) concentrations required for the panels 3. Develop data network within and between projects 4. Data-driven hypothesis: determine next phase priorities Experimental Requirements:    started

5. Establish Experimental Goal Tissue Sample Preparation Separation Method(s) Protein Identification (quantification, characterization) Subproteome Isolation Intrinsic Characteristics Mass (size) pI (charge) Hydrophobicity (oil/water solubility) Biospecificity (protein interactions) Validation in Larger Cohort Image Analysis (quantification) The Broad-based Proteomic Process Pam

6. Tissue Whole cell Cytoplasmic Myofilament Mitochondria Inner mitochondrial Membrane proteins Integral membrane N-linked glyco. Lipid rafts Peptide separation Shotgun-like (mudpit, 2DLC) Protein separation 1DE 1DLC (reversed phase) 2DLC (pI/hydrophobicity) 2DE (pI/mass) 2DLC + whole mass Isoelectric fractionation + 1DE or 2DE Quantification ITRAQ labeling (MS) O 16 /O 18 labeling (MS) Densitometry Broad based Discovery Review Graham et al., J Physiol. 2005

7. Each proteomic method has limitations in what types of proteins observed and information obtained The technical goal is to use correct combination of methods to maximize proteome coverage while balancing sample requirements, costs and resources

8. Work Flow Myofilament – 2DE, 1DLC Soluble – isoelectric fractionation + 2DE, 2DLC Mitochondria – 1DLC, 2DLC, 2DE Membrane protein – 1DLC, 2DLC N-linked glyco – pull down and 1DLC Myofilament – 2DE, 1DLC Soluble – isoelectric fractionation + 2DE, 2DLC Mitochondria – 1DLC, 2DLC, 2DE Membrane protein – 1DLC, 2DLC N-linked glyco – pull down and 1DLC

9. 98 of 200 protein spots identified: 79 non-redundant 20 proteins with PTM (pI shifts) MW Inner mitochondrial subproteome (IMM) 79 non-redundant with 20 PTM proteins (pI shifts) including novel phosphorylation of beta and alpha ATP synthase pH 4-7 pH 6-11

10. Beckman 2DLC intact protein separation pI and hydrophobicity First dimensionSecond dimension F2 Image analysis (developed Ludesi and JVE) Dried down, neutralize and enzymatic digest ESI MS MS (± quantification) 3 mg of IMM 224 proteins identified (146 non-redundant)

11. Kratos Axima CFR Comet macromizer™ 500 kDa 0 20 kDa 100 kDa Tracking all three intrinsic protein properties – pI, hydrophobicity coupled with whole mass MALDI-TOF MS Two instruments to expand mass range Stanley et al., Biomarkers 2005 Bob Cotter, JHU

12. 10 30 50 70 90 mass (kDa) AXIMA MALDI TOF Macromizer MALDI TOF Protein Theoretical mass pI Nuclease sensitive element binding protein 1 35,822 9.98 Endothelial differentiation-related factor 1 16,359 9.99 Ribosomal protein L38 8,213 10.10

13. Little protein overlap between three protein separation methods (total 401 proteins in IMM – 50 novel) 17 Multiple technologies increases proteome coverage and allows isoform and PTM determination

14. Validation and Focused approach i) Reversed arrays (proposed project) Spot whole tissue and various subproteomes onto slide Probe with Ab to protein and the phospho-protein Very powerful for signaling systems already established for JUNK, ERK, AKT in other cell types Nucleus Mitochondria Soluble extract Anti-AKT Ab Anti–phospho-AKT Ab NORMAL HF

15. ii) CHIP nano-western blots By Shimadzu and Proteome Systems Multiple western blots simultaneously (pL droplets) - Myofilament CHIP: Myosin, hUNC, phospho-hUNC, MBPC, phospho-MBPC, desmin, phospho-desmin, cTnT, phospho- cTnT, cTnI, PKA phospho-cTnI, compared with a generic phospho-AB anti-ATPase α Ab

16. Next Steps A)Single site sample preparation for broad based and focused approaches B)Broad based analysis i.Dunn – cytoplasmic analysis, myofilament ii.Van Eyk – mitochondria, membrane protein C)Validations i.Group - Selection of key candidate proteins from previous proteomic and genomic work ii.Dos Reemedios (Rodney) – test Abs for cardiac specificity and develop panels iii.Van Eyk/Dos Reemedios - analysis by CHIP (hUNC, CHIP, myosin, TnT ± PO 4- -2, TnI± PO 4- -2, MBPC ± PO 4- -2 D)Signaling pathways i.Petricoin/Dos Reemedios – reversed arrays for signaling pathway activation (selected pathways)

17. D) Build up accessible data base linking genomic, proteomic and functional data Note: an overlapping study could be carried out focused on the molecular changes due to aging. A huge thanks to Chris and his group for these Remarkable and priceless samples. A true visionary