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…