Curt T. Reimann

Terbutaline Enantiomer Separation and Quantification by Complexation and Field Asymmetric Ion Mobility Spectrometry-Tandem Mass Spectrometry.

Recently, we introduced a new approach to chiral separation and analysis of amino acids by chiral complexation and electrospray high-field asymmetric waveform ion mobility spectrometry coupled to mass spectrometry (ESI-FAIMS-MS). In the present work, we extended this approach to the separation of the drug compound terbutaline. Terbutaline enantiomers were complexed with metal ions and an amino acid to form diastereomeric complexes of the type [M(II)(L-Ref)2((+)/(-)-A)-H](+), where M(II) is a divalent metal ion, L-Ref is an amino acid in its L-form, and A is the terbutaline analyte. When metal and reference compound were suitably chosen, these complexes were separable by FAIMS. We also detected and characterized larger clusters that were transmitted at distinct FAIMS compensation voltages (CV), disturbing data analysis by disintegrating after the FAIMS separation and forming complexes of the same composition [M(II)(L-Ref)2((+)/(-)-A)-H](+), thus giving rise to additional peaks in the FAIMS CV spectra. This undesired phenomenon could be largely avoided by adjusting the mass spectrometer skimmer voltages in such a way that said larger clusters remained intact. In the quantitative part of the present work, we achieved a limit of detection of 0.10% (-)-terbutaline in a sample of (+)-terbutaline. The limit of detection and analysis time per sample compared favorably to literature values for chiral terbutaline separation by HPLC and CE.

Direct electron transfer reactions between human ceruloplasmin and electrodes.

In an effort to find conditions favouring bioelectrocatalytic reduction of oxygen by surface-immobilised human ceruloplasmin (Cp), direct electron transfer (DET) reactions between Cp and an extended range of surfaces were considered. Exploiting advances in surface nanotechnology, bare and carbon-nanotube-modified spectrographic graphite electrodes as well as bare, thiol- and gold-nanoparticle-modified gold electrodes were considered, and ellipsometry provided clues as to the amount and form of adsorbed Cp. DET was studied under different conditions by cyclic voltammetry and chronoamperometry. Two Faradaic processes with midpoint potentials of about 400 mV and 700 mV vs. NHE, corresponding to the redox transformation of copper sites of Cp, were clearly observed. In spite of the significant amount of Cp adsorbed on the electrode surfaces, as well as the quite fast DET reactions between the redox enzyme and electrodes, bioelectrocatalytic reduction of oxygen by immobilised Cp was never registered. The bioelectrocatalytic inertness of this complex multi-functional redox enzyme interacting with a variety of surfaces might be associated with a very complex mechanism of intramolecular electron transfer involving a kinetic trapping behaviour.

Relaxation dynamics of biopolymers seeded with unfolded lysozyme transients in vacuo. The role of primary sequence in protein folding

The availability of experimental data on biomolecular ions diffusing in a low-pressure gas has raised a number of important questions about the folding behaviour of anhydrous proteins in acuo. In this work, we explore an important aspect of the folding mechanism for anhydrous proteins, namely, its sensitivity to changes in primary sequence. To this end, we study the computer-simulated relaxation dynamics of protein conformers that share the same initial unfolded backbone geometry, but that differ in the primary sequences. The initial unfolded (transient) conformers are derived from an in acuo unfolding run of lysozyme. The relaxation behaviour of unfolded disulfide-intact lysozyme is compared with that of four other different sequences threaded to the same unfolded backbone geometry: disulfide-reduced lysozyme, cytochrome c′, polyglycine and polyalanine. Using a large ensemble of molecular dynamics trajectories, we monitor configurational transitions in a two-dimensional space of order parameters that convey changes in compactness and chain entanglement. Our results indicate that both disulfide-intact and disulfide-reduced lysozyme relax to structures with quasi-native compactness and entanglement. However, fast refolding appears to be more efficient in the presence of the disulfide bridges, since noncompact intermediates persist longer in disulfide-reduced lysozyme. The cytochrome c′ sequence threaded onto the lysozyme transient shows similar relaxation behaviour to that of disulfide-intact lysozyme. Yet, the cytochrome c′ sequence gives rise to several long-lived intermediates, one of which displays global molecular shape features similar to those of native cytochrome c′. In contrast, the relaxation of the polyglycine transient exhibits no initial large-scale collapse, but rather resembles the “pearling” transition of homopolymers (i.e., the initial formation of small locally compact blobs of chain). Polyalanine displays an intermediate behaviour, characterized by instances of both successful and frustrated global collapse. These findings shed light on how primary sequence affects specifically the formation of initial, persistent folding intermediates in acuo.

Role of electrostatic and van der Waals interactions on the in vacuo unfolding dynamics of lysozyme ions

Role of electrostatic and van der Waals interactions on the in vacuo unfolding dynamics of lysozyme ions