Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells

Pauliina M. Uitto, Braddon K. Lance, Graham R. Wood, James Sherman, Mark S. Baker, Mark P. Molloy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

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 languageEnglish
Pages (from-to)2105-2112
Number of pages8
JournalJournal of Proteome Research
Volume6
Issue number6
DOIs
Publication statusPublished - Jun 2007

Keywords

  • metabolic labeling
  • SILAC
  • two-dimensional gel electrophoresis
  • image analysis
  • MALDI-TOF/TOF
  • urokinase plasminogen activator
  • protein quantitation

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