Thomas Wyttenbach

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinsons disease and Alzheimers disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimers disease.

Effect of the long-range potential on ion mobility measurements

The temperature dependence of ion mobilities in helium was studied by using the ion chromatography method to investigate the effect of long-range terms in the ion-buffer gas interaction. Experimental cross sections thus determined increased significantly as the temperature was lowered from 300 to 80 K for all ions investigated, which were fullerene C60+, cationized PEG polymers, cationized crown ethers, and protonated and sodiated oligoglycines. The temperature dependence of the collision cross sections was successfully modeled by employing simple atom-atom interaction potentials including a repulsive R−12 term and the attractive long-range R−6 and R−4 terms, R being the distance between the colliding particles.

Ion mobility–mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation

Amyloid cascades that lead to peptide β-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with cellular machinery. The location and cause of the transformation from a natively unstructured assembly to the β-sheet oligomers found in all fibrils is important in understanding disease onset and the development of therapeutic agents. Largely, research on this early oligomeric region was unsuccessful because all the traditional techniques measure only the average oligomer properties of the ensemble. We utilized ion-mobility methods to deduce the peptide self-assembly mechanism and examined a series of amyloid-forming peptides clipped from larger peptides or proteins associated with disease. We provide unambiguous evidence for structural transitions in each of these fibril-forming peptide systems and establish the potential of this method for the development of therapeutic agents and drug evaluation.

Design of a new electrospray ion mobility mass spectrometer

Abstract The design of a new ion mobility mass spectrometer is presented. The design features an electrospray ion source; an ion funnel to transmit ions efficiently from the source to the mobility cell and to accumulate ions in the pulsed ion mode; a mobility cell, and a quadrupole mass analyzer. Each part of the instrument is described in detail. Preliminary results obtained with the new instrument are presented to demonstrate its capabilities. Equilibrium experiments showed that the Δ G °(300 K) values for the addition of the first water molecule to the doubly protonated peptides bradykinin, angiotensin II, and LHRH are in the range from −3.5 to −2.5 kcal/mol. The corresponding values for the singly protonated ions are >−0.5 kcal/mol for angiotensin II and LHRH, but equal to −2.6 kcal/mol for bradykinin. The stronger bonding in bradykinin may be due to the presence of a salt bridge structure. Ion arrival time distributions showed that singly protonated peptides can form aggregates of the form ( n M + n H) n + . The mobilities of these ions indicated that they are near spherical. Heating the drift cell to ∼450 K caused dissociation of the (2M + 2H) 2+ ion into two (M + H) + units on the 1 ms experimental time scale. A theoretical fit to the experimental data yielded rate constants and a barrier for dissociation of 30 ± 2 kcal/mol for bradykinin and 39 ± 3 kcal/mol for LHRH.

Structural Stability from Solution to the Gas Phase: Native Solution Structure of Ubiquitin Survives Analysis in a Solvent-Free Ion Mobility–Mass Spectrometry Environment

The conformations of desolvated ubiquitin ions, lifted into the gas phase by electrospray ionization (ESI), were characterized by ion mobility spectrometry (IMS) and compared to the solution structures they originated from. The IMS instrument combining a two-meter helium drift tube with a quadrupole time-of-flight mass spectrometer was built in-house. Solutions stabilizing the native state of ubiquitin yielded essentially one family of tightly folded desolvated ubiquitin structures with a cross section matching the size of the native state (1000 Å(2)). Solutions favoring the A state yielded several well-defined families of significantly unfolded conformations (1800-2000 Å(2)) matching in size conformations between the A state and a fully unfolded state. On the basis of these results and a wealth of data available in the literature, we conclude that the native state of ubiquitin is preserved in the transition from solution to the desolvated state during the ESI process and survives for >100 ms in a 294 K solvent-free environment. The A state, however, is charged more extensively than the native state during ESI and decays more rapidly following ESI. A state ions unfold on a time scale equal to or shorter than the experiment (≤50 ms) to more extended structures.

Amyloid β-protein monomer structure: A computational and experimental study

The structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimers disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Aβ42 peptide conformations are dominated by loops and turns but show some helical structure in the C‐terminal hydrophobic tail. A second calculation on Aβ42 in a solvent‐free environment yields compact structures turned “inside out” from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Aβ42 −3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross‐sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent‐free family of structures. Implications of the calculations on the early stages of aggregation of Aβ42 are discussed.

Gas-Phase Conformations: The Ion Mobility/Ion Chromatography Method

Ion mobility spectrometry coupled to mass spectrometry provides a powerful tool to explore the three-dimensional shape of polyatomic ions. Applications include the investigation of cluster ion geometries and conformations of flexible molecules such as biopolymers and synthetic polymers. The ion structure is obtained by measuring collision cross sections in a high pressure drift tube filled with helium and comparing it to model structures obtained by various theoretical methods such as molecular modeling and electronic structure calculations. The temperature of the drift tube is generally adjustable (typically from 80 to 800 K) providing a unique opportunity to address topics such as the thermal motion of floppy molecules, the unfolding process of folded structures, the kinetics of structural interconversion, and the kinetics of dissociation processes. In addition, the ion mobility instrumentation can be used to obtain thermochemical data of ligand addition reactions, giving important additional information about the polyatomic ions under investigation. The theoretical background and the concepts of these ion mobility based experiments and the instrumentation employed are briefly reviewed in this chapter. Furthermore, some detailed examples and a very brief summary of selected applications found in the literature are given.

Inclusion of a MALDI ion source in the ion chromatography technique: conformational information on polymer and biomolecular ions

Abstract A matrix-assisted laser desorption ionization (MALDI) source has been coupled to the ion chromatography instrument developed at UCSB. The source produces a strong, consistent signal for several hours on a single sample. In this paper we report the application of this method to a series of poly(ethylene glycol) (PEG) polymers cationized by sodium. Data have been taken for Na + PEG5 to Na + PEG19. The temperature dependence of the ion mobility (collision cross-section) in He gas for Na + PEG9, Na + PEG13 and Na + PEG17 has been measured from 80 to 580 K. A detailed analysis of these three systems has been accomplished in order to extract the conformations of the ion and how they vary with temperature. This analysis included several significant changes from methods used previously. Molecular mechanics methods were used both to obtain the lowest energy 0 K structures and to predict how these structures would change as temperature increases. In order to account for the observed low temperature results, a 12-6-4 potential was incorporated in place of the hard-sphere potential used previously. For all three systems studied in detail, the oxygen atoms on the PEG units solvated the Na + ion, forming a crown ether type ring of five oxygens surrounding Na + and several others above and below this ring. The molecular mechanics model was also applied to neutral PEG13. In this instance a quite compact structure is obtained for T ≤ 200 K but a sudden melting type transition occurs between 200 and 300 K and chaotic motion dominates at and above 300 K. Data are also reported on the temperature dependence of the ion mobility of C 60 + . This ion is expected to change shape only slightly over the temperature range reported here. Consequently it provided an excellent set of calibration data for evaluating the intramolecular interaction potentials used to describe the collision process.

Gas phase conformations of biological molecules: the hydrogen/deuterium exchange mechanism

A model was developed to describe the deuterium uptake of gas phase polypeptide ions via H/D exchange with D2O. Ab initio calculations established, for energetic reasons, that the exchange must take place via a “relay” mechanism involving both a charged site and a nearby basic site. Molecular dynamics simulations indicated that the D2O molecule did not penetrate the core of the example peptide, protonated bradykinin (Bk+H)+, and hence the relay mechanism must occur on the peptide surface. Two factors were deemed to be important: (1) The surface accessibility of the charged sites and the basic sites and (2) the distances between them. An algorithm was developed that accounted for these features using the absolute exchange rate as a free parameter. Excellent agreement was obtained with experiment when equal weight was given to an ensemble of low energy conformations of (Bk+H)+, assumed to have a salt bridge primary structure. Single conformations, or other protonated forms, did not allow good agreement with experiment for any value of the absolute exchange rate constant.

Ion Mobility Analysis of Molecular Dynamics

The combination of mass spectrometry and ion mobility spectrometry (IMS) employing a temperature-variable drift cell or a drift tube divided into sections to make IMS-IMS experiments possible allows information to be obtained about the molecular dynamics of polyatomic ions in the absence of a solvent. The experiments allow the investigation of structural changes of both activated and native ion populations on a timescale of 1-100 ms. Five different systems representing small and large, polar and nonpolar molecules, as well as noncovalent assemblies, are discussed in detail: a dinucleotide, a sodiated polyethylene glycol chain, the peptide bradykinin, the protein ubiquitin, and two types of peptide oligomers. Barriers to conformational interconversion can be obtained in favorable cases. In other cases, solution-like native structures can be observed, but care must be taken in the experimental protocols. The power of theoretical modeling is demonstrated.