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Experiment Design for iTRAQ Validation

Experiment Design for iTRAQ Validation. Rob McMaster, Gabriela Cohen-Freue, Robert Balshaw, Axel Bergman, Raymond Ng and others in the Biomarker Team (Vancouver, Victoria). Assessment of the Proteome.

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Experiment Design for iTRAQ Validation

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

  2. 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

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

  4. Plasma Biomarker Discovery: an Application using iTRAQ Each depleted plasma sample is digested with trypsin All 4 samples are pooled Quantitative 2DLC / MS/MS analysis Protein identification and differential expression analysis Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  5. 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

  6. Plasma pool composed of 16 healthy individuals. CONTROL Plasma depletion (9x) Bloc B Pool of 3 depletions Bloc A Pool of 3 depletion Bloc C Pool of 3 depletions A2 A4 B2 B4 C2 C4 A1 A3 B1 B3 C1 C3 Validation Experimental Design Experiment replicates Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  7. GenomeBC Proteomic Centre iTRAQ Validation of Experiment Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  8. 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

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

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

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

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

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

  14. 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

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

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

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

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

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

  20. 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

  21. GenomeBC Proteomic Centre iTRAQ Validation of Experiment Depleted control plasma + S0 = 1x b-gal S1 = 1x b-gal S2 = 2x b-gal S3 = 10x b-gal Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

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

  23. 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

  24. 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 Biomarkers in TransplantationDiscovery Strategy: Proteomics Analysis Plasma Jack Bell Research Centre, Dr. Robert McMaster BioLibrary ITRAQ Analysis UVic Genome BC Proteomics Platform Victoria, BC Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  25. Venn Diagram of Identified Proteins A B 8 5 10 88 1 3 3 C Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

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

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