title: Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells.
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 (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 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.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, 2109, Sydney, NSW, Australia.
authors.forename: Pauliina M
authors.initials: PM
authors.lastname: Uitto
authors.forename: Braddon K
authors.initials: BK
authors.lastname: Lance
authors.forename: Graham R
authors.initials: GR
authors.lastname: Wood
authors.forename: James
authors.initials: J
authors.lastname: Sherman
authors.forename: Mark S
authors.initials: MS
authors.lastname: Baker
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
mesh_terms: D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D007553:Isotope Labeling; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000201:enzymology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D009363:Neoplasm Proteins; D014568:Urokinase-Type Plasminogen Activator