Differentiation of Compact and Extended Conformations of Di-Ubiquitin Conjugates with Lysine-Specific Isopeptide Linkages by Ion Mobility-Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry 2011-08, Vol.22 (8), p.1463-1471
• Main Authors: Jung, Ji Eun, Pierson, Nicholas A., Marquardt, Andreas, Scheffner, Martin, Przybylski, Michael, Clemmer, David E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Aminoacids, peptides. Hormones. Neuropeptides; Analytical Chemistry; Analytical, structural and metabolic biochemistry; Bacteria; Bioinformatics; Biological and medical sciences; Biotechnology; Cellular structure; Chemical synthesis; Chemistry; Chemistry and Materials Science; Circular Dichroism; Conjugates; Cyclotron resonance; Dichroism; Differentiation; Elongation; Exact sciences and technology; Fourier transforms; Fundamental and applied biological sciences. Psychology; Humans; Ionic mobility; Ions; Linkages; Lysine; Lysine - chemistry; Lysine - metabolism; Mass spectrometry; Mathematical models; Modelling; Models, Molecular; Molecular Sequence Data; Organic Chemistry; Polypeptides; Proteins; Proteomics; Reactivity and mechanisms; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Research Article; Scientific imaging; Spectroscopy; Tandem Mass Spectrometry - methods; Ubiquitins - chemistry; Ubiquitins - metabolism
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-011-0158-0

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.