title: Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.
abstract: The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: Lucia
authors.initials: L
authors.lastname: Geis-Asteggiante
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: Emma K
authors.initials: EK
authors.lastname: Dixon
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: Meredith
authors.initials: M
authors.lastname: Miller
authors.affiliation: Proteomics Core Facility, University of Maryland, College Park, MD, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive University of Maryland, College Park, MD, 20742, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D007536:Isomerism; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000032:analysis* / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D025801:Ubiquitin
keywords: branched proteins; electron transfer dissociation; graphical software; linkage sites; tetraubiquitin; top-down analysis; topology; tribrid orbitrap