In this thesis, the application of electrospray ionisation mass spectrometry (ESI-MS) to the analysis of intact proteins is demonstrated. In papers I and II, the use of ESI-MS for the analysis of noncovalent protein-ligand complexes were discussed. In addition, the interfacing of liquid chromatography (LC) with ESI-MS and the development of an LC-ESI-MS method were demonstrated in paper III for the quality control of recombinant proteins. Furthermore, this method was applied in paper IV for the analysis of covalent glycosyl-enzyme intermediates. The monitoring of noncovalent complexes by ESI-MS is well established. However, the varying characteristic of ESI-MS data, especially in the analysis of noncovalent complexes can make the quantification of such complexes troublesome. In paper I, it was demonstrated how the variation in the position of the ESI-emitter and the initial droplet size of the electrosprayed droplets, together with different partitioning of a protein and its ligand in these droplets, can be the cause of such varying characteristics. Furthermore, it was shown that the partitioning can be of electrostatic and/or hydrophobic/hydrophilic origin. Thus it was demonstrated that if the ligand is more hydrophobic and thereby more surface active relative to the protein, decreasing the droplet size or increasing the distance between the electrospray emitter and the sampling orifice will lead to more efficient sampling of the droplet bulk where the ligand concentration is low. This results in a favoured sampling of free protein relative to the protein ligand complex. The opposite was shown to occur if the ligand is more hydrophilic than the protein. In paper II, Ribonuclease A (RNAse) was used as a model for enzymes acting on polymeric substrates with different chain lengths. Nano-ESI-MS was applied to monitor the noncovalent interactions between RNAse and different target ligands. Among the single building blocks of RNA, including ribose, the bases adenine, guanine, cytosine and uracil, and phosphate, only phosphate was observed to interact at multiple RNAse sites at a higher cone voltage. Furthermore, monobasic singlestranded deoxycytidylic acid oligomers (dCx) of different lengths (X=6, 9 and 12), and RNAse were analysed with nano-ESI-MS. The deoxycytidylic acid with 12 nucleotides was observed with the highest complex to free protein ratio, hence indicating the strongest interaction. Finally, collision induced dissociation of the noncovalent RNAseA-dC6 complex resulted in dissociation of covalently bound cytosine from the nucleotide backbone rather than break up of the noncovalent complex, illustrating the cooperative effect of multiple noncovalent interactions. In paper III an LC-ESI-MS method was presented capable of analysing proteins 10-100 kDa in size, from salt-containing liquid samples. The proteins included human protein fragments for the largescale production of antibodies and human protein targets for structural determination, expressed in E. coli. Also, glycosylated proteins expressed in Pichia pastoris were analysed. The method provides fast chromatography, is robust and makes use of cheap desalting/trap columns. In addition it was used with optimised reduction and alkylation protocols in order to minimize protein aggregation of denatured and incorrectly folded proteins containing cysteins, which otherwise form adducts by disulfide bond formation. Furthermore, the method was used in paper IV for the quantification of covalent proteinligand intermediates formed enzymatically between PttXET16-34, a xyloglucan endo-transglycosylase (XET) from hybrid aspen, and the synthetic substrates GalGXXXGGG and GalXXXGXXXG designed in order to function as donor substrates only. Thus covalent GalG-enzyme and GalGXXXG-enzyme complexes were detected. Moreover, establishing of a pseudo equilibrium for the formation of the covalent GalGXXXG-enzyme complex enabled quantification of the saccharide and enzyme constituents of this equilibrium and determination of the free energy of formation (∆G0). The high mass resolution of the TOF-MS allowed unambiguous assessment of the covalent nature of the glycosyl-enzyme complexes. Morover, the formation of noncovalent complexes between excess substrate and protein, which can deteriorate MS-signal and increase spectrum complexity, was efficiently avoided by the chromatographic step, which separated the saccharide content from the protein content. / QC 20100913

Die Dissertation besteht aus zwei Teilen: im ersten Teil wird das Verhalten von kleinen Edelgasclustern in intensiven Laserfeldern theoretisch untersucht. Im zweiten Teil wenden wir die Bohmsche Mechanik auf die Untersuchung von Helium und des Wasserstoffmolkülions (H_2^+) in intensiven Pulsen an. Im ersten Teil wird zunaechst ein numerisches Modell entwickelt, welches es erlaubt, unter Mitnahme aller Elektronen die die Dynamik kleiner Edelgascluster in starken Feldern zu simulieren. Anschliessend wird detailliert untersucht, wie die Expansion eines Clusters Einfluss auf dessen Absorptionseigenschaften nimmt. Wir verallgemeinern dabei den aus der Molekuelphysik bekannten "enhanced-ionization" Mechanismus auf den Bereich der Clusterphysik. Im zweiten Teil wird die in der Bohmschen Mechanik gegebene Moeglichkeit einer mikroskopischen Untersuchung der Wellenfunktionsdynamik verwendet, um atomphysikalische Prozesse unter einem neuen Aspekt zu betrachten. Der Ionisationsprozess des Wasserstoffmolekuelions im starken Lichtfeld wird eingehend untersucht, insbesondere die Frage, bei welchen Kernabstaenden die Ionisation stattfindet. Fuer das Heliumatom liefert die Analyse der zur Einfach- bzw. Doppelionisation fuehrenden Anfangszustaende der Bohmschen Testteilchen neue Einblicke in das nichtsequentielle Ionisationsverhalten.

Um Proteine und Peptide ohne Verluste in der Trennleistung mit Hilfe der Kapillarelektrophorese zu analysieren, wurde der Innenkanal von fused silica Kapillaren mit 2-Hydroxyethylmethacrylat (HEMA) nach dem Prinzip der radikalischen Atomtransferpolymerisation beschichtet. Durch die Variation einiger Reaktionsparameter konnten dabei vier HEMA-Beschichtungen erhalten werden. Mit diesen wurden dann nach den Methoden der Kapillarzonenelektrophorese bei pH 3 und pH 9 und der isoelektrischen Fokussierung Vergleichsmessungen durchgeführt. Die Detektion erfolgte dabei teilweise mit einem Elektrosprayionisations-Flugzeit-Massenspektrometer. Es stellte sich heraus, dass sich die entwickelten Kapillarbeschichtungen im Bezug auf Trenneffizienz wie auch Stabilität im basischen Bereich teilweise stark voneinander unterschieden. Die Betrachtung der Polymerschichten mit Hilfe der Atomkraftmikroskopie zeigte, dass die Morphologie der HEMA-Beschichtung stark vom gewählten Reaktionsmedium abhängig ist.

Les différents mécanismes d'ionisation dissociative consécutifs à la collision entre un ion projectile multichargé (S15+ 13.6 MeV/u) et la molécule D2, ont été étudiés à partir des corrélations vectorielles entre les vecteurs impulsions de fragments D+ et d'électrons produits. Le travail a donc dans un premier temps consisté à développer un dispositif expérimental capable de détecter en coïncidence toutes les particules chargées produites lors d'une collision. La mesure de leur vecteur impulsion, qui permet d'accéder à leur énergie cinétique et leur direction d'émission initiale par rapport à celle du faisceau, combine les techniques de temps de vol et de localisation. La corrélation entre les énergies cinétiques des fragments et des électrons produits permet d'une part de déterminer la contribution de chacun des mécanismes d'ionisation dissociative au processus global d'ionisation, et d'autre part de séparer sans ambiguïté les distributions d'énergie cinétique des fragments D+ correspondant à chacun de ces mécanismes. Enfin, l'analyse spatiale des corrélations vectorielles conduit aux distributions angulaires d'émission électronique par rapport à la direction du faisceau, pour une orientation de l'axe de la molécule et une énergie de l'électron données. Les distributions mesurées sont comparées à celles obtenues par le modèle théorique CDW-EIS.

Reproduction de : Thèse de doctorat : Physique nucléaire : Lyon 1 : 2006. / Titre provenant de l'écran titre. 101 réf. bibliogr.

For the first time, a full-dimensional quantum-mechanical description of excitation, dissociation and ionization of H2+ in intense laser fields is presented. The quantum-mechanical propagation of the nuclei is carried out approximately using time-dependent Floquet surfaces and the Coulomb surface, switching between these surfaces is possible stochastically (”hopping”). The impact of quantum effects in the nuclear dynamics on dissociation and ionization as well as their interplay is investigated in detail. The results are in excellent agreement with experimental data. It is shown in particular that quantum effects in the nuclear dynamics are essential for the description and interpretation of the experiments.

This thesis presents an extensive computational study of electron-impact scattering and ionisation of atomic hydrogen and hydrogenic ions, which are fundamental to many diverse disciplines, from astrophysics and nuclear fusion to atmospheric physics. The non-relativistic Schrodinger equation describes these collisions, though finding solutions for even hydrogen, the simplest electron-atom collision, has proven to be a monumental task. Recently, Rescigno et al [Science 286, 2474 (1999)] solved this equation in coordinate space using exterior complex scaling (ECS), and presented the first electron-hydrogen differential cross sections for ionisation that matched with experiment without requiring uncontrolled approximation. This method has significant potential for extension to larger collision systems, but its large computational demand has limited its energy range and target configurations, and its application to discrete final-state collisions has been largely unexplored. Using radically different numerical algorithms, this thesis develops methods that improve the computational efficiency of ECS by two orders of magnitude. It extends the method to calculate discrete final-state scattering cross sections and enhances the target description to include hydrogenic ions and excited initial states. In combination, these developments allow accurate solutions over a broad range of energies and targets, for both scattering and ionisation, including the important near-threshold energy region where accurate calculations have been unavailable. The refined ECS method implemented in this work now offers complete numerical solutions of electron-hydrogen collisions, and its computational efficiency will facilitate its future application to more complex targets. The thesis culminates with the first ab initio quantum mechanical confirmation of ionisation threshold laws for electron-hydrogen collisions [Bartlett and Stelbovics, 2004, Phys. Rev. Lett. 93, 233201], which have resisted confirmation through the complete solution of the Schrodinger equation for more than half a century.

University, Diss., 2007--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

München, Techn. Univ., Diss., 2003.

Techn. Universiẗat, Diss., 2003--Berlin.