title: Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry.
abstract: Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.
authors: Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada.
authors: Nicholas J
authors: NJ
authors: Denis
authors: Julian
authors: J
authors: Vasilescu
authors: Jean-Philippe
authors: JP
authors: Lambert
authors: Jeffrey C
authors: JC
authors: Smith
authors: Daniel
authors: D
authors: Figeys
mesh_terms: D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D012015:Reference Standards; D014357:Trypsin / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D014357:Trypsin