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

114115116117

114

115

116 117

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…