Comparative Proteome Analysis of Ovarian Epithelial Cell Lines for Cancer Biomarker Discovery
This study compares proteomic profiles of two human epithelial ovarian cell lines, TOV-81D (low malignant) and TOV-112D (highly aggressive), to identify potential cancer biomarkers. Using traditional 2D gel electrophoresis and modern iTRAQ labeling techniques, researchers analyzed protein expression variations. The results highlighted the strengths and limitations of each method, revealing 65 proteins identified via 2D GE and 37 via iTRAQ, with 10 proteins overlapping. Both methods showed promise in the search for biomarkers, emphasizing the complexity of proteomic studies in cancer research.
Comparative Proteome Analysis of Ovarian Epithelial Cell Lines for Cancer Biomarker Discovery
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Presentation Transcript
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 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)
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
Advantages: • Well established technique • Visualization of the protein spots • Detection of modified proteins Disadvantages: • Laborious and time consuming • MW cut-off (6 – 250 kDa)
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)
“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
Analysis of iTRAQ-labeled Peptides • 114 • 115 • 116 • 117 • 116 • 117 • 115 • 114
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
Final Score: • 2D GE/LC-MS – 65 (51) proteins • iTRAQ LC-MS – 37 (32) proteins • Crossover – 10 proteins
Conclusions: • Both approaches were successful in the biomarker search • Each approach has specific advantages and limitations • There is no easy way for proteomics studies…
