Vlad, Camelia

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Vlad
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Camelia
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Online saw-bioaffinity-mass spectrometry : new tool for simultaneous detection, structure determination and affinity quantification of protein-ligand interactions from biological material

2014, Slamnoiu, Stefan, Stumbaum, Mihaela, Vlad, Camelia, Lindner, Kathrin, Karreman, Christiaan, Leist, Marcel, Przybylski, Michael

Bioaffinity analysis using biosensors such as surface plasmon resonance has become an established technique for the detection and quantification of biomolecular interactions. However, a principal limitation of biosensors is their lack of providing structure analysis of affinity-bound ligands. We have developed an online combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the simultaneous structure determination and affinity quantification of biopolymer ligands, dissociated from the biopolymer- ligand complex on a gold chip. Key tool of the SAW-MS combination is an interface that provides sample concentration and in-situ desalting for the MS analysis of the ligand eluate. Recent applications of the online bioaffinity- MS show broad bioanalytical potential for direct interaction studies from biological material, as diverse as antigen-antibody and lectin- carbohydrate complexes; affinity binding constants (KD) are determined from milli- to nanomolar ranges [1,2]. Moreover, we report here first applications of the online- SAW- MS to the direct top-down structural characterization of proteolytic intermediates and oligomers in the aggregation of Parkinson’s Disease key protein, alpha-synuclein (αSyn) [3] from brain homogenate.

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Proteolytic intermediates in the oligomerisation-aggregation pathway of alpha-synuclein revealed by ion mobility mass spectrometry

2010, Przybylski, Michael, Vlad, Camelia, Lindner, Kathrin, Ciossek, Thomas, Hengerer, Bastian, Leist, Marcel

A variety of diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by the formation of ”misfolded”protein aggregates. While ”soft-ionisation”mass spectrometry (MS), particularly electrospray-MS (ESI-MS), has substantially contributed to peptide analysis and proteomics, ESI-MS is not suitable to direct ”in-situ”analysis of conformational states and intermediates. Recently, ion mobility mass spectrometry (IM-MS) is emerging as a new tool to probe protein structures and interactions due to its potential for separation polypeptides by conformational states, shape and topology. We report here first applications of IM-MS to the characterization of reaction intermediates in the in vitro oligomerisation and aggregation of alpha-synuclein (αSyn), a key polypeptide in Parkinson’s disease. IM-MS of the in vitro aggregation of wt-αSyn enabled the structure elucidation of several hitherto unknown N- and C-terminal products, and a proteolytic fragment at V71-T72 in the aggregation domain (C-VT72; 7.2 kDa), which appears to be a key intermediate in the aggregation pathway; in vitro studies of this fragment prepared by chemical synthesis and bacterial expression showed a dramatically enhanced rate of aggregation. Most recently, IM-MS was also successfully applied to the direct analysis of affinity-captured αSyn from biological samples, such as brain homogenate, indicating this method as a powerful new tool to the molecular characterization of conformation-dependant intermediates of protein aggregation.

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Fragmentation and aggregation of physiological and parkinson- synucleins revealed by ion mobility and affinity- mass spectrometry

2014, Przybylski, Michael, Slamnoiu, Stefan, Stumbaum, Mihaela, Vlad, Camelia, Lindner, Kathrin, Karreman, Christiaan, Kipping, Marc, Desor, M., Leist, Marcel, Hengerer, Bastian

A large variety of cellular processes are based on the formation of supramolecular protein assemblies. Several diseases, previously thought to be unrelated, are characterised by “misfolded” protein aggregates. Structures and reaction pathways of pathophysiological aggregates are only poorly characterised at present. “Soft-ionisation” mass spectrometry (MS), such as HPLC-MS, is often unsuitable to the analysis of reactions and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) is emerging as a powerful tool for analysis of protein aggregation due to its concentration-independent gas phase separation capability. Applications of IM-MS to the in vitro oligomerization- aggregation of α-synuclein (αSyn), a key protein for Parkinson’s disease, provided the first identification of a specific autoproteolytic fragmentation, particularly a highly aggregation-prone fragment by cleavage at the triplett, VVT(70-72) of the central aggregation domain [1]. The corresponding recombinant αSyn(72-140) fragment showed substantially faster aggregation and high neurotoxicity compared to the intact protein. The recent development of online bioaffinity-MS [2] enabled first direct structural studies in vivo, e.g. from brain homogenate. Applications of affinity-MS will be discussed for the characterization of oligomers and assemblies in vivo. Moreover, specific mutations of the central (70-72) triplett in synucleins provided a breakthrough by mutation of VFS(70-72) from ß-Synuclein into the αSyn sequence, that completely abolished neurotoxic aggregation. These results suggest ion mobility- MS and affinity-MS as powerful tools for the elucidation of structures and intermediates of polypeptide aggregation, providing a basis for the molecular study of oligomerization- aggregation pathways, and the design of specific inhibitors of aggregation.

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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.