Przybylski, Michael

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Ion Mobility- Mass Spectrometry and Affinity- Mass Spectrometry : New Tools for elucidation of structures and reaction pathways of "misfolding" - aggregating neurodegenerative proteins

2012, Przybylski, Michael, Lindner, Kathrin, Vlad, Camelia, Manea, Marilena, Pierson, Nicholas A., Strube, I., Karreman, Christiaan, Schildknecht, Stefan, Leist, Marcel, Rontree, John

A large variety of cellular processes are based on the formation and dynamics of multi- and supramolecular protein assemblies, and several diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by “misfolded” protein aggregates. Chemical structures and reaction pathways of pathophysiological aggregates are only poorly characterised and understood at present. “Softionisation” mass spectrometry (MS), such as HPLC-electrospray-MS, is often unsuitable to direct analysis of reaction pathways and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) has been emerging as a new tool for analysis of protein aggregates due to its concentration-independent gas phase separation capability. First applications of IM-MS to in vitro oligomerization products of α-synuclein (αSyn) and ß amyloid key proteins for Parkinson’s disease and Alzheimer’s disease, enabled hitherto unknown truncation and aggregation products to be identified. Studies of the in vitro oligomerization- aggregation of αSyn provided the first identification of specific autoproteolytic fragments, previously observed by gel electrophoresis but not identified [1]. A highly aggregating fragment found by cleavage at V71- T72 in the central aggregation domain of αSyn, αSyn(72-140), was prepared by chemical synthesis and recombinant expression and showed substantially faster oligomerization- aggregation, and higher neurotoxicity compared to the intact protein. Recently, the development and application of combined (online) affinity- MS methods enabled the structural identification of epitope-specific Aß-antibodies that disaggregate Aßplaques, and physiological neuroprotective Aß-autoantibodies inhibiting the formation of Aßaggregates. These results indicate ion mobility- MS and affinity- MS as powerful tools for the molecular elucidation of structures and intermediates of polypeptide aggregates. The structures thus obtained provide a basis for (i), the detailed study of oligomerization- aggregation pathways; (ii), the design of peptides capable of inhibiting or modifying aggregation; and (iii), the evaluation of new immunotherapeutic lead structures.

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Functional Ubiquitin Conjugates with Lysine-epsilon-Amino-Specific Linkage by Thioether Ligation of Cysteinyl-Ubiquitin Peptide Building Blocks

2009, Jung, Ji Eun, Wollscheid, Hans-Peter, Marquardt, Andreas, Manea, Marilena, Scheffner, Martin, Przybylski, Michael

The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-ε-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the "donor" component, with a corresponding lysine-ε-amino-branched haloacyl-activated ubiquitin "acceptor" peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1-52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54-76)2. Advantages of the present approach are as follows: (i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.