iTRAQ

iTRAQ

The Centre has developed and validated more than 40 SOPs for the iTRAQ procedure employing both LC-ESI-QTOF and LC-MALDI-TOF/TOF. Based on these SOPs coefficients of variation of less than 20% are achievable as demonstrated in a manuscript recently submitted for publication. The SOPs are currently extended for the use of the 8-plex iTRAQ reagents. Please visit Biomarker Discovery and Validation for additional information in iTRAQ.

The Centre is developing approaches in which iTRAQ is combined with specific enrichment strategies, including enrichment for phospho-peptides, to quantify changes in the modification level of proteins. Furthermore, the Centre develops approaches and techniques to improve the sensitivity of iTRAQ analysis. This includes specific sample handling steps to reduce the sample amount required and the implementation of exclusion lists in order to mine further down into the proteome. These exclusion lists are created from the first analytical round of data, which will be applied as a filter for re-analysis of the remaining sample. Previous experiments with the use of exclusion lists have resulted in the identification of approximately 50% more proteins than in the initial analyses.

Selected Proteomics Centre iTRAQ Publications
  1. Kuzyk MA, Ohlund LB, Elliott MH, Smith D, Qian H, Delaney A, Hunter CL, Borchers CH. A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-QqTOF and MALDI-TOF/TOF mass spectrometers. Proteomics. 5;9(12):3328-3340 (2009).
  2. Lippert DN, Ralph SG, Phillips M, White R, Smith D, Hardie D, Gershenzon J, Ritland K, Borchers CH, Bohlmann J. Quantitative iTRAQ Proteome and Comparative Transcriptome Analysis of Elicitor-Induced Norway Spruce (Picea abies) Cells Reveals Elements of Calcium Signaling in the Early Conifer Defense Response. Proteomics. 9(2):350-67 (2009).
  3. Lippert DN, Ralph SG, Phillips M, White R, Smith D, Hardie D, Gershenzon J, Ritland K, Borchers CH, Bohlmann J. Quantitative iTRAQ Proteome and Comparative Transcriptome Analysis of Elicitor-Induced Norway Spruce (Picea abies) Cells Reveals Elements of Calcium Signaling in the Early Conifer Defense Response. Proteomics. 9(2):350-67 (2009).
  4. Rosenzweig D, Smith D, Myler PJ, Olafson RW, Zilberstein D. Post-translational modification of cellular proteins during Leishmania donovani differentiation. Proteomics. 8(9):1843-50 (2008).
  5. Rosenzweig D, Smith D, Opperdoes F, Stern S, Olafson RW, Zilberstein D. Retooling Leishmania metabolism: from sand fly gut to human macrophage. Journal of the Federation of American Societies for Experimental Biology. 22(2): 590-602 (2008).
  6. Lucker J, Laszczak M, Smith D, Lund ST. Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation. BMC Genomics. Jan 26;10:50. (2009).