title: A COFRADIC protocol to study protein ubiquitination.
abstract: Here, we apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and to identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation, after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). Because USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the ε-amino group of the ubiquitinated lysine, this enzyme reintroduces primary ε-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. This method led to the identification of over 7500 endogenous ubiquitination sites in more than 3300 different proteins in a native human Jurkat cell lysate.
authors.affiliation: Department of Medical Protein Research, VIB , B-9000 Ghent, Belgium.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Stes
authors.forename: Mathias
authors.initials: M
authors.lastname: Laga
authors.forename: Alan
authors.initials: A
authors.lastname: Walton
authors.forename: Noortje
authors.initials: N
authors.lastname: Samyn
authors.forename: Evy
authors.initials: E
authors.lastname: Timmerman
authors.forename: Ive
authors.initials: I
authors.lastname: De Smet
authors.forename: Sofie
authors.initials: S
authors.lastname: Goormachtig
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D010449:Peptide Mapping; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D057149:Ubiquitinated Proteins / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D020543:Proteome; D057149:Ubiquitinated Proteins