title: Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry.
abstract: The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25,600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top-down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive, 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, 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, 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, College Park, MD, 20742, USA.
authors.forename: Yeji
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive, College Park, MD, 20742, USA.
authors.forename: Tanuja R
authors.initials: TR
authors.lastname: Kashyap
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, 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, College Park, MD, 20742, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000595:Amino Acid Sequence; D025821:Polyubiquitin / Q000737:chemistry; D055503:Protein Multimerization; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin
keywords: branched proteins; electron transfer dissociation; polyubiquitins; top-down analysis; workflow