Marquardt, Andreas


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Differentiation of compact and extended conformations of di-ubiquitin conjugates with lysine-specific isopeptide linkages by ion mobility-mass spectrometry

2011-08, Jung, Ji Eun, Pierson, Nicholas A., Marquardt, Andreas, Scheffner, Martin, Przybylski, Michael, Clemmer, David E.

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.

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Identification, affinity characterisation and biological interactions of lectin-like peptide-carbohydrate complexes derived from human TNF-alpha using high-resolution mass spectrometry

2007, Marquardt, Andreas, Bernevic, Bogdan, Przybylski, Michael

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-α [TNF-α (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-α protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.

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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.


Mass spectrometric approaches for elucidation of antigen antibody recognition structures in molecular immunology

2007, Stefanescu, Raluca, Iacob, Roxana Elena, Damoc, Eugen, Marquardt, Andreas, Amstalden, Erika, Manea, Marilena, Perdivara, Irina, Maftei, Madalina, Paraschiv, Gabriela-Ioana, Przybylski, Michael

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, affinity proteomics using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a β- amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer s disease. Using an immobilized antigen and antibody- proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; parexprot ). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of molecular antibody-recognition signatures offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.