Better Biomarkers in Transplantation. A Genome Canada Initiative for Human HealthImmunity & Infection Research Centre

Rob McMaster, Gabriela Cohen-Freue, Robert Balshaw, Axel Bergman, Raymond Ng and others in the

Biomarker Team (Vancouver, Victoria)

Experiment Design for iTRAQ Validation

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Assessment of the Proteome• Identifying and quantitating the proteome is one

key approach to understanding their roles in biology, and at times in health

• One specific strategy relies on mass spectrometry, wherein the various proteins are examined on the basis of mass and charge

• One of the techniques currently used in this regard is ITRAQ, wherein ratios of amounts of different proteins are obtained between control and experimental samples

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Labeling(Applied Biosystems)

+ Peptide

14

14

1212

16

14

14

1313

16

14

15

1313

17

15

15

1313

18

114

115

116

117

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Each depleted plasma sample is digested with trypsin

Quantitative 2DLC / MS/MS analysis

Protein identification and differential expression analysis

Each digested sample is labeled with an iTRAQ reagent

1 Pooled plasma control sample iTRAQ reagent 114

2 Baseline (Plasma just before transplant) iTRAQ reagent 115

3 Week 1 after the transplant iTRAQ reagent 116

4 Week 2 – acute rejection identified iTRAQ reagent 117

All 4 samples are pooled

Plasma Biomarker Discovery: an Application using iTRAQ

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Validation Experiment

Key Questions:

• How reproducible is the iTRAQ procedure?

• Is there a channel-specific bias?

• How reproducible is the quantitation with respect to the concentrations of a specific protein?

• How similar are the depletion runs with respect to the proteins that are retained?

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Validation Experimental Design

Plasma pool composed of 16 healthy individuals.

CONTROL

Plasma depletion (9x)

Bloc APool of 3 depletion

Bloc BPool of 3 depletions

Bloc CPool of 3 depletions

A1

A2

A3

A4

B1

B2

B3

B4

C1

C2

C3

C4

Experiment replicates

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Experimental bloc A iTRAQ Label

Depletion 1

Rep # 114 115 116 117

A1 S0 S1 S2 S3

A2 S1 S2 S3 S0

A3 S2 S3 S0 S1

A4 S3 S0 S1 S2

Experimental bloc B iTRAQ Label

Depletion 2

Rep # 114 115 116 117

B1 S0 S2 S3 S1

B2 S2 S3 S1 S0

B3 S3 S1 S0 S2

B4 S1 S0 S2 S3

Experimental bloc C iTRAQ Label

Depletion 3

Rep # 114 115 116 117

C1 S0 S3 S1 S2

C2 S3 S1 S2 S0

C3 S1 S2 S0 S3

C4 S2 S0 S3 S1

GenomeBC Proteomic Centre iTRAQ Validation of Experiment

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Validation Experiment

Key Questions:

• How reproducible is the iTRAQ procedure?

• Is there a channel-specific bias?

• How reproducible is the quantitation with respect to the concentrations of a specific protein?

• How similar are the depletion runs wrt the proteins that are retained?

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Comparing replications: Row analysisProtein x

Re

lativ

e a

bu

nd

an

ce

A1 A2 A3 A4

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Within Variability

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Distribution of means: Not Normalized (NN)

A1 A2 A3 A4

0.5

1.0

1.5

2.0

2.5

3.0

Block A

A B C B4

0.5

1.0

1.5

2.0

2.5

3.0

Block B

C1 C2 C3 C4

0.5

1.0

1.5

2.0

2.5

3.0

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Distribution of means: Normalized (N)

A1 A2 A3 A4

0.5

1.0

1.5

2.0

2.5

3.0

Block A

B1 B2 B3 B4

0.5

1.0

1.5

2.0

2.5

3.0

Block B

C1 C2 C3 C4

0.5

1.0

1.5

2.0

2.5

3.0

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Standard Deviations and Within variability: NN

A1 A2 A3 A4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block A

B1 B2 B3 B4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block B

C1 C2 C3 C4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Standard Deviations and Within variability: NThe Effect of Normalization

A1 A2 A3 A4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block A

B1 B2 B3 B4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block B

C1 C2 C3 C4 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Validation Experiment

Key Questions:

• How reproducible is the iTRAQ procedure?

• Is there a channel-specific bias?

• How reproducible is the quantitation with respect to the concentrations of a specific protein?

• How similar are the depletion runs wrt the proteins that are retained?

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Comparing Ratios: column analysisProtein x

Re

lativ

e a

bu

nd

an

ce

115/114 116/114 117/114

0.6

0.7

0.8

0.9

1.0

1.1

1.2

MeanRatio117MeanRatio115

MeanRatio116

Total Mean

Variance Ratio117

Variance Ratio116

Variance Ratio115

Within Variability

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Distribution of means: NN

115/114 116/114 117/114

0.5

1.0

1.5

2.0

2.5

Block A

115/114 116/114 117/114

0.5

1.0

1.5

2.0

2.5

Block B

115/114 116/114 117/114

0.5

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Distribution of means: N

115/114 116/114 117/114

1.0

1.5

2.0

2.5

Block A

115/114 116/114 117/114

1.0

1.5

2.0

2.5

Block B

115/114 116/114 117/114

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Standard Deviations and Within variability: NN

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block A

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block B

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Standard Deviations and Within variability: N

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block A

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block B

115/114 116/114 117/114 Average

0.0

0.5

1.0

1.5

2.0

2.5

Block C

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Validation Experiment

Key Questions:

• How reproducible is the iTRAQ procedure?

• Is there a channel-specific bias?

• How reproducible is the quantitation with respect to the concentrations of a specific protein?

• How similar are the depletion runs wrt the proteins that are retained?

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Experimental bloc A iTRAQ Label

Depletion 1

Rep # 114 115 116 117

A1 S0 S1 S2 S3

A2 S1 S2 S3 S0

A3 S2 S3 S0 S1

A4 S3 S0 S1 S2

Experimental bloc B iTRAQ Label

Depletion 2

Rep # 114 115 116 117

B1 S0 S2 S3 S1

B2 S2 S3 S1 S0

B3 S3 S1 S0 S2

B4 S1 S0 S2 S3

Experimental bloc C iTRAQ Label

Depletion 3

Rep # 114 115 116 117

C1 S0 S3 S1 S2

C2 S3 S1 S2 S0

C3 S1 S2 S0 S3

C4 S2 S0 S3 S1

Depleted control plasma +S0 = 1x -galS1 = 1x -galS2 = 2x -galS3 = 10x -gal

GenomeBC Proteomic Centre iTRAQ Validation of Experiment

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Ra

tio

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

S1(1x) S2(2x) S3(10x)

Block A

Block B

Block C

Mean

Reproducibility and Quantitation of added -galactosidase

-galactosidase

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

iTRAQ Validation Experiment

Key Questions:

• How reproducible is the iTRAQ procedure?

• Is there a channel-specific bias?

• How reproducible is the quantitation with respect to the concentrations of a specific protein?

• How similar are the depletion runs with respect to the proteins that are retained?

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

BioLibraryPlasma

Jack Bell Research Centre, Dr. Robert McMaster

Depleted PlasmaColumn 1: albumin, fibrinogen, IgG, IgA, IgM, a1-antitrypsin, transferrin, haptoglobin, a1-acid glycoprotein , HDL Apolipoprotein A-I, HDL Apolipoprotein A-II, a2-macroglobulinColumn 2: Apolipoprotein B, Complement C3

ITRAQ AnalysisUVic Genome BC

Proteomics PlatformVictoria, BC

Biomarkers in TransplantationDiscovery Strategy: Proteomics Analysis

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Venn Diagram of Identified Proteins

C

B

10

3

5888

1 3

A

Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

Summary

• Platform validation is critical to assess the severity of various bias – The proposed experimental design may be applicable

to other platforms

• Also important to determine various platform parameters– Between and within run experimental variation is

approx. 15% to 20%– Counting false positives (>2 fold-change) we estimate

approx 0.3% false positive rate

• Statistical analyses continue…