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

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

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

Representative 2D gel images for the two ovarian cell lines

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…