1. Immunity & Infection Research Centre Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Rob McMaster, Gabriela Cohen-Freue, Robert Balshaw, Axel Bergman, Raymond Ng and others in the Biomarker Team (Vancouver, Victoria) Experiment Design for iTRAQ Validation

2. 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

3. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health iTRAQ Labeling (Applied Biosystems) + Peptide 14 12 16 14 13 16 14 15 13 17 15 13 18 114 115 116 117

4. 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 1Pooled plasma control sampleiTRAQ reagent  114 2Baseline (Plasma just before transplant) iTRAQ reagent  115 3Week 1 after the transplantiTRAQ reagent  116 4Week 2 – acute rejection identifiediTRAQ reagent  117 All 4 samples are pooled Plasma Biomarker Discovery: an Application using iTRAQ

5. 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?

6. 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 A Pool of 3 depletion Bloc B Pool of 3 depletions Bloc C Pool of 3 depletions A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 Experiment replicates

7. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Experimental bloc A iTRAQ Label Depletion 1 Rep #114115116117 A1S0S1S2S3 A2S1S2S3S0 A3S2S3S0S1 A4S3S0S1S2 Experimental bloc BiTRAQ Label Depletion 2 Rep #114115116117 B1S0S2S3S1 B2S2S3S1S0 B3S3S1S0S2 B4S1S0S2S3 Experimental bloc CiTRAQ Label Depletion 3 Rep #114115116117 C1S0S3S1S2 C2S3S1S2S0 C3S1S2S0S3 C4S2S0S3S1 GenomeBC Proteomic Centre iTRAQ Validation of Experiment

8. 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?

9. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Comparing replications: Row analysis Within Variability

10. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Distribution of means: Not Normalized (NN)

11. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Distribution of means: Normalized (N)

12. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Standard Deviations and Within variability: NN

13. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Standard Deviations and Within variability: N The Effect of Normalization

14. 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?

15. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Comparing Ratios: column analysis MeanRatio117 MeanRatio115 MeanRatio116 Total Mean Variance Ratio117 Variance Ratio116 Variance Ratio115 Within Variability

16. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Distribution of means: NN

17. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Distribution of means: N

18. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Standard Deviations and Within variability: NN

19. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Standard Deviations and Within variability: N

20. 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?

21. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Experimental bloc A iTRAQ Label Depletion 1 Rep #114115116117 A1S0S1S2S3 A2S1S2S3S0 A3S2S3S0S1 A4S3S0S1S2 Experimental bloc BiTRAQ Label Depletion 2 Rep #114115116117 B1S0S2S3S1 B2S2S3S1S0 B3S3S1S0S2 B4S1S0S2S3 Experimental bloc CiTRAQ Label Depletion 3 Rep #114115116117 C1S0S3S1S2 C2S3S1S2S0 C3S1S2S0S3 C4S2S0S3S1 Depleted control plasma + S0 = 1x  -gal S1 = 1x  -gal S2 = 2x  -gal S3 = 10x  -gal GenomeBC Proteomic Centre iTRAQ Validation of Experiment

22. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Ratio Reproducibility and Quantitation of added  -galactosidase  -galactosidase

23. 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?

24. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health BioLibrary Plasma Jack Bell Research Centre, Dr. Robert McMaster Depleted Plasma Column 1: albumin, fibrinogen, IgG, IgA, IgM, a1-antitrypsin, transferrin, haptoglobin, a1-acid glycoprotein, HDL Apolipoprotein A-I, HDL Apolipoprotein A-II, a2-macroglobulin Column 2: Apolipoprotein B, Complement C3 ITRAQ Analysis UVic Genome BC Proteomics Platform Victoria, BC Biomarkers in Transplantation Discovery Strategy: Proteomics Analysis

25. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health Venn Diagram of Identified Proteins C B 10 3 5 8 88 13 A

26. 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…