Gas‐phase protein separation by ion mobility: With its ability to separate the Parkinson's disease protein α‐synuclein and its autoproteolytic products—despite the small concentrations of the latter—ion‐mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization‐aggregation. In particular, a possible key fragment, the highly aggregating C‐terminal fragment, αSyn(72–140), has been revealed.

The antimycotic drug clotrimazole inhibits the function of the gastric H,K-ATPase in a manner similar to that observed for the Na,K-ATPase. Because of the high hydrophobicity of the compound, the interaction between clotrimazole and the ion pump occurs at the membrane domain in the apolar core of the membrane. The enzymatic activity was inhibited with a half-saturating concentration of 5.2 microM. Various partial reactions of the pump cycle were analyzed with the electrochromic styryl dye RH421 that has been widely used to study the transport mechanism of P-type ATPases. We discovered that the interaction of clotrimazole with the H,K-ATPase introduces a single "dead-end" branch added to the Post-Albers scheme in the E(1) state of the pump. In this inhibiting state, the ion binding sites have a significantly enhanced affinity for protons and bind up to two protons even at pH 8.5. Inhibition of the pump can be reversed by a decreased pH or increased K(+) concentrations. The mechanistic proposal that allows an explanation of all experiments presented is similar to that published for the Na,K-ATPase.

Abstract. Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions.Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural ({"' characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn forMS of the ligand eluate from the biosensor. Suitable MSacquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF-MS systems. Applications are presented for mass spectrometric identifications and affinity (K 0 ) determinations of the neurodegenerative polypeptides, r..-amyloid (AI1), and pathophysiological and physiological synucleins (a-and r..-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of aSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

The inside cover picture shows the first molecular identification of autoproteolytic fragments of the Parkinson's disease protein a-synuclein, in particular, the highly aggregation-prone fragment (72-140). On p. 2740 ff, M. Przybylski et al. explain how the gas-phase separation capability of ion-mobility mass spectrometry allowed these fragments to be identified. Inside CoverCamelia Vlad, Kathrin Lindner, Christiaan Karreman, Stefan Schildknecht, Marcel Leist, Nick Tomczyk, John Rontree, James Langridge, Karin Danzer, Thomas Ciossek, Alina Petre, Michael L. Gross, Bastian Hengerer, and Michael Przybylski* The inside cover picture shows the first molecular identification of autoproteolytic fragments of the Parkinson's disease protein a-synuclein, in particular, the highly aggregation-prone fragment (72-140). On p. 2740 ff, M. Przybylski et al. explain how the gasphase separation capability of ion-mobility mass spectrometry allowed these fragments to be identified.