The World of BiomarkersProteomics: Molecular Dissection

Update on collaboration between

Cris Dos Reemedios

Jenny Van Eyk

Mike Dunn

Chip Petricoin

Goal

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

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 levelsCorrelate to functional data

Validate potential candidate proteins (and pathways) Carry out further functional analysis of key pathways Develop new hypothesis

1. In-depth analysisa) 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 analysisa) 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

started

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-basedProteomic Process

Pam

Pam

Tissue• Whole cell• Cytoplasmic• Myofilament• Mitochondria• Inner mitochondrial • Membrane proteins• Integral membrane• N-linked glyco. • Lipid rafts

Peptide separationShotgun-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)• O16/O18 labeling (MS)• Densitometry

Broad basedDiscovery

Review Graham et al., J Physiol. 2005

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

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

98 of 200 protein spots identified: 79 non-redundant20 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

Beckman 2DLC intact protein separation pI and hydrophobicity

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8.5UV-1SMP-3.0mg-020304

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UV-2SMP-3.0mg-020304_02

First dimension Second dimension F2

Image analysis (developed Ludesi and JVE)

Dried down, neutralize and enzymatic digest

ESI MS MS (± quantification)

3 mg of IMM224 proteins identified(146 non-redundant)

Kratos Axima CFR

Comet macromizer™

500 kDa0 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 2005Bob Cotter, JHU

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

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

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

NucleusMitochondriaSoluble extract

Anti-AKT Ab Anti–phospho-AKT Ab

NORMAL

HF

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

Next StepsA) Single site sample preparation for broad based and focused

approaches

B) Broad based analysisi. Dunn – cytoplasmic analysis, myofilamentii. Van Eyk – mitochondria, membrane protein

C) Validationsi. Group - Selection of key candidate proteins from previous proteomic

and genomic workii. Dos Reemedios (Rodney) – test Abs for cardiac specificity and develop

panelsiii. Van Eyk/Dos Reemedios - analysis by CHIP (hUNC, CHIP, myosin,

TnT ± PO4--2 , TnI± PO4-

-2, MBPC ± PO4--2

D) Signaling pathwaysi. Petricoin/Dos Reemedios – reversed arrays for signaling pathway

activation (selected pathways)

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 theseRemarkable and priceless samples.

A true visionary