Abstract
Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (∼8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV ∼25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty.
Original language | English |
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Pages (from-to) | 2105-2112 |
Number of pages | 8 |
Journal | Journal of Proteome Research |
Volume | 6 |
Issue number | 6 |
DOIs | |
Publication status | Published - Jun 2007 |
Keywords
- metabolic labeling
- SILAC
- two-dimensional gel electrophoresis
- image analysis
- MALDI-TOF/TOF
- urokinase plasminogen activator
- protein quantitation