Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry.
Post-translational modification of proteins via the covalent attachment of Ubiquitin (Ub) plays an important role in the regulation of protein stability and function in eukaryotic cells. In the present study, we describe a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We have demonstrated the applicability of this approach by identifying 70 ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. This method will aid the study of protein ubiquitination and may be used as a tool for the discovery of novel biomarkers that are associated with disease progression.
Vasilescu
Julian
J
Ottawa Institute of Systems Biology, Ontario, Canada.
Smith
Jeffrey C
JC
Ethier
Martin
M
Figeys
Daniel
D
D014408:Biomarkers, Tumor; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D061988:Proteasome Inhibitors; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry*
D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D020543:Proteome; D025801:Ubiquitin; D014357:Trypsin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde