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Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions

Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions


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STUMBAUM, Mihaela, 2014. Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions

@phdthesis{Stumbaum2014Devel-26503, title={Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions}, year={2014}, author={Stumbaum, Mihaela}, address={Konstanz}, school={Universität Konstanz} }

Stumbaum, Mihaela 2014-02-24T06:18:19Z Stumbaum, Mihaela The development of new hybrid analytical methods comprising the significant recent advances of mass spectrometry (MS), promotes the versatility of MS applications in life science research. In particular, electrospray mass spectrometry (ESI-MS) has emerged as a powerful technique for analysing intact gas phase ions from large biomolecules and their complexes. However, in contrast to the already large number of ESI-mass spectrometric studies of protein structures and their modifications, mass spectrometric chacterisation and kinetic evaluation of interactions of biological complexes by ESI-MS has not yet been possible. The development of methods that combine biosensor and mass spectrometric structural characterisation of peptides and proteins with the kinetic characterisation of biopolymer complex formation was the main goal of this thesis.<br /><br /><br />The present thesis is divided into five parts. A first part focused on the development of a new method for the simultaneous mass spectrometric structural characterisation and the kinetic evaluation of protein- ligand interactions. Both an ESI- Ion trap mass spectrometer and a high resolution Fourier transform-ion cyclotron resonance-mass spectrometer (ESI-FTICR-MS) were successfully coupled with a Surface Acoustic Wave (SAW) biosensor. The interface developed for the online coupling of SAW with ESI-MS utilises a six-port valve micro-column and micro-injector for desalting and in-situ concentration of proteins after dissociated from the biopolymer complex. A second interface was developed that provides automatic switching of the two valves for transfer into the mass spectrometer. Several parameters were optimised including flow rates, buffers, and electrospray conditions for the ionisation process. The online SAW-ESI-MS approach was successfully validated and applied to tyrosine-nitrated peptides interacting with anti-3-nitrotyrosine (3-NT) antibodies and Lysozyme interacting with an anti- Lysozyme antibody.<br /><br /><br />The second part of this thesis was dealing with the identification of the epitope of tyrosine-nitrated peptides recognised by specific anti-3-NT antibodies. Tyrosine and nitro-tyrosine containing peptides from different protein sequences been identified to contain nitration sites in vivo and in vitro, were synthesised by solid-phase peptide synthesis. 3-NT was used with standard Fmoc and side-chain protection chemistry (t-butyl, trityl) and showed high coupling yields of crude peptides. The synthetic peptides were characterised by mass spectrometry and used in immuno-analytical studies. ESI-MS provided unambiguous identification of 3-NT containing peptides by the characteristic 45 Da mass shifts corresponding to the addition of NO2+. In contrast, UV-MALDI-MS of 3-NT containing peptides showed photochemical fragmentation at the nitro-phenyl group which hampers of impedes the unequivocal identification of tyrosine nitrations. Complemented by conventional immunoanalytical techniques (Dot blot, ELISA), affinity-mass spectrometry proved to be an efficient tool in the characterisation of recognition specificities of a monoclonal anti 3-NT antibody.<br /><br /><br />Affinity-MS, ELISA, and online SAW-ESI-MS revealed that the epitope depends not only on the nitrated tyrosine residue, but requires positively charged amino acids (Lys and/or Arg) in close proximity to the 3-NT residue. In contrast, neutral or negatively charged residues showed decreased binding affinity. The anti 3-NT antibody binding to tyrosine-nitrated Prostacyclin Synthase (PCS) peptides, as well as nitrated peptides derived from Eosinophil proteins (ECP, EDN and EPO), showed a similar motif with adjacent basic amino acids. These results suggest structural orientation of the nitrated tyrosine on the protein surface, and stabilising effect by surrounding positively charged amino acids. The capability of anti-3-NT antibodies to discriminate between nitro-tyrosine in different environments in proteins should be useful for producing antibodies to specific motifs and for the development of specific biomarkers.<br /><br /><br />Further interaction studies of the peptides binding to anti-3-NT antibodies were performed using a new methodology, hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of antibodies for peptides. This method, based on a previously established method of protein-ligand interaction study by mass spectrometry, titration, and H/D exchange (PLIMSTEX), makes use of a dilution strategy (dPLIMSTEX), whereby the mass of the peptide ligand is the readout. Using this method, two monoclonal anti-3-NT antibodies were determined to have low nanomolar binding affinities to three nitrated PCS peptides. Additionally, the binding stoichiometry was determined.<br /><br /><br />The third part of the dissertation was devoted to applications of SAW-ESI-MS for the detection, structural identification and quantification of peptides bound to the calcium (Ca2+)-binding protein Calmodulin (CaM) complexed with Ca2+. The interaction of Calmodulin with enzymes is inhibited by a number of pharmacological agents and small basic polypeptides that share common structural features. Although the exact nature of calmodulin-enzyme interactions is not clear, a likely hypothesis is that Ca2+-binding to calmodulin exposes hydrophobic clefts which may be important in the binding of inhibitors and target enzymes.<br /><br />Interaction studies of CaM with two peptides, Melittin and Substance P, were performed by online SAW-ESI-MS, and kinetic determinations performed together with the MS identification. Determination of Substance P-CaM and Mellitin-CaM association and dissociation kinetics was performed by extracting the data from the sensor signals for all concentrations employed, using the 1:1 binding model, and provided KD values of 44 nM for CaM-Substance P, and 16 nM for CaM-Mellitin.<br /><br /><br />A fourth part of the dissertation describes the SAW based detection of interactions of nucleotide transcription factor with Ets peptides. The Ets family of transcriptions factors is fundamental to development and homeostatic processes including DNA replication and repair, cell growth and division and apoptosis. A common feature of Ets proteins is a conserved 85 amino acid domain that binds specifically to double stranded DNA (dsDNA) containing a (G/A)(A/C)GGAAGT consensus sequence. Kinetic analysis of dsDNA binding Ets1 provided a low nanomolar KD value. Further quantification of affinity-bound transcription factor derived peptides to dsDNA was performed and showed different results from those obtained using a peptide microarray. The differences in the binding results can be explained by difference in the experimental setup between the two technologies employed. In the peptide microarray, a library of peptides is synthesised directly on 384-microwell plates and binding of the dsDNA is monitored by fluorescent detection. In the SAW technique, the dsDNA is covalently immobilised on the surface, and the binding of the various peptides is determined. The differing results by both technologies suggest that the conformation of the peptides is crucial for binding to dsDNA. Thus, in the microarray approach, the conformations of the peptide may be different from that of the peptides in solution, thus further studies may be required in order to understand these differences.<br /><br /><br />In the final part of this thesis, affinity studies RA33 peptides, specific for Rheumatoid Arthritis to a monoclonal anti-RA33 antibody, and autoantibody patient sera were performed. RA33, a major autoantigen in RA patients and anti-RA33 specific antibodies often appear shortly after the onset of RA. A previously identified RA33 epitope was characterised by SAW biosensor using a monoclonal anti-RA antibody and a KD in the low nanomolar range was determined. Further interaction studies of RA33 derived peptides were performed with RA patient sera samples. The peptides were covalently immobilised on a carboxyl surface and the interaction of 1:200 patient sera determined by the SAW biosensor. The peptide identified in the interaction with the monoclonal anti-RA33 antibody was found to have the highest affinity to the patient samples. Bioaffinity measurements performed using the SAW technology showed to be highly sensitive in the detection of the autoantibodies present in patient sera. eng deposit-license 2014 Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions 2014-02-24T06:18:19Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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