title: Differentiation of compact and extended conformations of di-ubiquitin conjugates with lysine-specific isopeptide linkages by ion mobility-mass spectrometry.
abstract: Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.
authors.affiliation: Laboratories of Analytical Chemistry and Biochemistry, and Graduate School, Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Jung
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Pierson
authors.forename: Andreas
authors.initials: A
authors.lastname: Marquardt
authors.forename: Martin
authors.initials: M
authors.lastname: Scheffner
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
authors.forename: David E
authors.initials: DE
authors.lastname: Clemmer
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014452:Ubiquitins; C075986:diubiquitin conjugate; D008239:Lysine