title: Insights into the molecular composition of endogenous unanchored polyubiquitin chains.
abstract: The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.
authors.affiliation: School of Biomedical Sciences, University of Nottingham, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Strachan
authors.forename: Lucy
authors.initials: L
authors.lastname: Roach
authors.forename: Kleitos
authors.initials: K
authors.lastname: Sokratous
authors.forename: David
authors.initials: D
authors.lastname: Tooth
authors.forename: Jed
authors.initials: J
authors.lastname: Long
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Garner
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D025821:Polyubiquitin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D053719:Tandem Mass Spectrometry; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D055767:Immobilized Proteins; D010446:Peptide Fragments; D057149:Ubiquitinated Proteins; D025821:Polyubiquitin; C000602677:TNFAIP3 protein, rat; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3