title: Preparing to read the ubiquitin code: a middle-out strategy for characterization of all lysine-linked diubiquitins.
abstract: Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)-based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all-native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS-based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp-selective acid cleavage, separation by reverse phase high-performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine-linked ubiquitin dimers.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Lee
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castañeda
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000595:Amino Acid Sequence; D008239:Lysine / Q000032:analysis*; D008872:Microwaves; D008969:Molecular Sequence Data; D025821:Polyubiquitin / Q000737:chemistry*; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014452:Ubiquitins / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D014452:Ubiquitins; C075986:diubiquitin conjugate; D025821:Polyubiquitin; D008239:Lysine
keywords: mass spectrometry; microwave-assisted acid cleavage; middle-out; proteomics; ubiquitin