Comparative Proteome Analysis Gagne et al. Proteome Sci. 2007.
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Transcript of 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
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