“ Panning for Biomarker Gold”

Applying Proteome Partitioning and

Fractionation to Biomarker Discovery

生物醫學部產品專員 劉聖德

美商貝克曼庫爾特有限公司台灣分公司

2007, 01, 16

Plasma is a Treasure Trove for Biomarker Discovery

Typical Protein Abundances in Human PlasmaL

og10

Con

cent

rati

on p

g/m

l

Anderson, N.L.and Anderson, N.G. Molecular and Cellular Proteomics, 1.11, 845-867 (2002)

12 Proteins Comprise up to 96% Total Plasma Protein

Partitioning instead of depletion

The Classic Problem for Biomarker Discovery

Partitioning Highly Abundant Proteins

• Partitioning Highly Abundant Proteins Enables:

– Proteome Enrichment – Improving detection limits

– Removal of “Masking” effect of Highly Abundant Proteins (Impact of HAP Peptide Mass Fingerprints on MS/MS based Protein ID)

• Multiplexed Immunoaffinity Strategy Most Ideal– IgG Based Methodology – IgY Based Methodology

IgY Immunoaffinity Reagents

• Polyclonal• Similar structure to IgG• Higher Avidity• Broader Antigen binding host range• Cleaner Capture

Avian Antibodies (Y – egg “yolk”)

Phylogenetic Differences of Albumin

• Significant differences between chicken & human

• Mammalian proteins highly immunogenic in birds

• Broad Antigen Binding Host Range

FASEB, 4, 2528-2532 (1990)

#’s represent % Amino Acid Similarity to Human Serum Albumin

Cleaner Capture

Capture Efficiency

• Formulate to antibody avidity and protein concentration level

• IgY ProteomeLab Solutions – Optimized for

– Primates (IgY 12, 12 protein partition highlighted above)

– Rodents (IgY R-7, 7 proteins optimized for mouse and rat)

Available in two configurations

IgY 12, IgY R-7

-- IgY-12 Spin Column (20ul blood)

-- IgY-12 LC 2 (6.4 x 63mm) (50ul blood)

-- IgY-12 LC10 (12.7 x 79mm)

(250ul blood)

–Selected Singles

•IgY Fibrinogen SC

•IgY HSA SC

•IgY RSA SC

•IgY Total IgG SC

•IgY HDL SC

ProteomeLab IgY Proteome Partitioning Kits

Minutes

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AU

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Three-step operation dilution stripping neutralization

flow through

bound

IgY Proteome Partitioning Chemistry – LC10

ProteomeLabTM PF 2DProteomeLabTM PF 2D

An Automated Two-Dimensional Protein Fractionation System

One Day, Automated, All Liquid

ProteomeLabTM PF 2D Flow Diagram

pHMonitor

UVDetector

CE/MS

MALDI

SELDI

ESI-MS

LC/MS

Microarray

ELISA

Injector

1st Dimension

Isoelectric Point

2nd Dimension

Hydrophobicity

Automated 2D proteome fractionation Strategy

Proteome Map

Liquid Phase Fractions

ProteomeLabTM PF 2D Method

Plasma Proteome Fractionation – PF 2D

3.0 mg plasma loaded

3.0 mg IgY-12 partitioned plasma

Differential Display

Red Bands – Predominate in PlasmaGreen Bands – Predominate in IgY-Partitioned Plasma

pI - hydrophobicity map - pH 7.3 – 7.6 (fract 19) highlighted

Hepatocyte Cell Culture

Data courtesy of Dr. David Lubman, University of Michigan

C. R. Middaugh et.al., Molecular & Cellular Proteomics, 2004, 3 (8), pp 746-769

Differential Display of COS-7 before (red) /after (green) Interaction with Heparin Sulfate

J82 Cells (bladder cell carcinoma cell lines)

Growth Matrix Effects on Phenotype

Hematoxylin and eosin labeled transverse sections of 3-dimensional J82 cultures grown on A) Matrigel , B) on plastic or C) on SISgel.

Dr. Robert Hurst, University of Oklahoma

Differential Display of SISgel Vs Matrigel

Comparison of cells grown on SISgel vs. Matrigel

Peak Differences Between Extracellular Matrices

Comparison 2nd Dimension Peak Absorbance Min pH Max pH Retention Time (min) Ratio

SISgel > Matrigel 4.03 4.33 13.66 30.504.03 4.33 15.36 9.035.54 5.84 17.67 3.225.84 6.13 15.69 5.005.84 6.13 15.81 4.757.32 7.62 16.67 9.328.06 8.06 13.59 315.558.06 8.06 16.01 60.60

Matrigel > SISgel 3.82 3.84 16.75 3.543.82 3.84 17.56 2.733.82 3.84 18.07 2.803.82 3.84 18.79 2.553.82 3.84 20.69 12.825.84 6.13 15.33 2.066.72 7.02 16.71 11.957.02 7.32 16.74 2.508.08 8.08 16.69 3.928.08 8.08 17.17 1.898.08 8.08 16.60 2.908.08 8.08 17.09 1.568.16 8.16 16.63 2.10

1st Dimension pH Range

Dr. Oliver Bogler, MD Anderson Cancer Center, Houston Tx

Comparison of pI/ hydrophobicity maps for cisplatin (red) and BBR3464 (green) treated gliomal cells, lane 18.

untreated cisplatin treated(tiosephosphate isomerase I)

HepG2 Cell Cycle analysis

Flow cytometry analysis of cell cycle using propidium iodide (PI) staining

Prof Paul Robinson, Purdue University

# cells sorted 1 x 106

Sorting Cells for PF 2D Fractionation

Dr. Jennifer Van Eyk, Johns Hopkins University

Orthogonal Approach – Information is Additive

Summary• PF 2D provides reproducible automated proteome based

fractionation by charge (pI) and hydrophobicity.

• Visualization maps allow you target change in a proteome for further study.

• Liquid fractionation allows you to maintain proteins in an intact state, allowing determination of post-translational modification.

• Removal of proteins of high abundance reduces Peptide Mass Fingerprints based masking of lower abundant proteins.

• IgY immunoaffinity technology allows cleaner capture across a broader host range.

• ProteomeLab IgY-12 Allows you to partition and remove up to 96% of the serum/plasma proteome enriching low abundant proteins.

Johns Hopkins University Jennifer Van Eyk

Purdue University J. Paul Robinson, Eli Asem

Oklahoma University Health Sciences Center, Oklahoma City Robert Hurst, Kimberly Kyker University of Michigan, Ann ArborDavid Lubman

GenWay Biotech, San Diego Wei-Wei Zhang, Jerry Feitelson, Xiangming Fang

University of Ilinois, Urbana Neil Kelleher, Andy Forbes, Mike Boyne

Beckman Coulter, Fullerton CA Michael Simonian, Edna Betgovargez, Jim Zhang, Ingred Cruzado Park

Acknowledgements