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Comparative Proteome Analysis

Comparative Proteome Analysis. Gagne et al. Proteome Sci. 2007. Main Goal: Compare proteomics for two human epithelial ovarian cell lines in search for cancer biomarkers. Morphology of the two human epithelial ovarian cell lines.

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Comparative Proteome Analysis

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  1. Comparative Proteome Analysis Gagne et al. Proteome Sci. 2007

  2. Main Goal: Compare proteomics for two human epithelial ovarian cell lines in search for cancer biomarkers

  3. Morphology of the two human epithelial ovarian cell lines TOV-81D cells (low malignant) show a flat morphology similar to normal human ovarian epithelium (A) TOV-112D cells (extremely aggressive) show a highly rounded morphology characteristic of highly transformed cell lines (B)

  4. Traditional 2D-GE Proteomics Technique • Obtain cell lysates • Run 2D-GE for each sample (in triplicates) • Compare the gels and spot over-expressed and/or under-expressed proteins • Identify those proteins (for each spot): • Cut the spot and trypsinaze the protein • Run LS-MS • Identify at least two unique peptides

  5. Advantages: • Well established technique • Visualization of the protein spots • Detection of modified proteins Disadvantages: • Laborious and time consuming • MW cut-off (6 – 250 kDa)

  6. Detection of carbonylated (oxidized) proteins A – 2D gel of total proteins from wild-type Arabidopsis seeds B – The indicated portion of the gel C, D, E – Revelation of carbonylated proteins with the anti-DNP immunoassay: C, Dry mature seeds; D, Seeds incubated in water; E, Seeds incubated with salicylic acid (Job et al., 2005; Rajjou et al., 2006)

  7. “Full digest” Proteomics Technique with iTRAQ Labeling • Obtain cell lysates • Trypsinaze each lysate • Label each lysate with one iTRAQ reagent • Combine samples (up to 4 lysates) • Perform pre-fractionation (IEF, IEC) • Run LC-MS (10-20 fractions) • Identify and simultaneously quantify peptides and parent proteins

  8. Analysis of iTRAQ-labeled Peptides • 114 • 115 • 116 • 117 • 116 • 117 • 115 • 114

  9. Advantages: • No 2D gels, no MW cut-off • Combine up to 4 digested samples • Reliable quantitation Disadvantages: • Complex peptide mixtures • Additional in vitro labeling step • Additional pre-fractionation step

  10. Representative 2D gel images for the two ovarian cell lines

  11. Final Score: • 2D GE/LC-MS – 65 (51) proteins • iTRAQ LC-MS – 37 (32) proteins • Crossover – 10 proteins

  12. Conclusions: • Both approaches were successful in the biomarker search • Each approach has specific advantages and limitations • There is no easy way for proteomics studies…

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