In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's 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 Alzheimer's disease.Many diseases share the common trait of peptide-protein misfolding that leads to oligomerization and, eventually, formation of plaques of β-sheet structure. Prominent among these are type 2 diabetes 1 , Parkinson's disease 2 and Alzheimer's disease 3,4 . Of these, Alzheimer's disease is the leading cause of late-life dementia and is the focus of this paper. An increasing body of evidence links oligomerization of a ubiquitous peptide, the amyloid-β [3][4][5][6] . For this reason, elucidation of pathways of oligomer formation may be critical for the identification of therapeutic targets.Many types of oligomeric amyloid-β assemblies have been described (for a review, see Lazo et al. 7 ). Recently, Bitan et al. [8][9][10] used photoinduced cross-linking of unmodified proteins (PICUP) to reveal that the 42-residue form of amyloid-β, Aβ42, formed (Aβ42) 5 and (Aβ42) 6 oligomers ('paranuclei') that could oligomerize to form structures of higher order. Aβ40 did not form paranuclei, but instead existed as a mixture of monomers, dimers, trimers and tetramers. Chen and Glabe 11 , in contrast, used fluorescence and gel electrophoresis to determine oligomer states of amyloid-β refolded from denaturing solutions. They observed only Aβ42 monomer and trimer bands, and no oligomers of Aβ40. Differences such as these may exist because of the diverse experimental systems used to monitor amyloid-β selfassociation. Also, it has been argued that, in addition to the intrinsic potential of amyloid-β to traverse different assembly pathways, flaws in experimental design may have misled researchers in their quest to elucidate fully the amyloid-β oligomerization process 12 . Hence there is significant uncertainty about amyloid-β oligomer states and their position and relevance to amyloid-β aggregation. Results and discussionWe used a different, more direct, method to probe the amyloid-β oligomerization process: ion mobility coupled with mass spectrometry [13][14][15] . Details are given in the Methods section.Here the results for Aβ40 are given as an example. The mass spectrum of Aβ40 is s...

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.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values ( K 0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E / N ; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method‐dependent results) only if the gas nature, temperature or E / N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40. Here, we apply mass spectrometry and ion mobility to the study of Abeta42 and its Pro19 alloform. The Phe19 --> Pro19 substitution blocks fibril formation by [Pro19]Abeta42. Evidence indicates that solution-like structures of Abeta monomers are electrosprayed and characterized. Unfiltered solutions of Abeta42 produce only monomers and large oligomers, whereas [Pro19]Abeta42 solutions produce abundant monomers, dimers, trimers, and tetramers but no large oligomers. When passed through a 10,000 amu filter and immediately sampled, Abeta42 solutions produce monomers, dimers, tetramers, hexamers, and an aggregate of two hexamers that may be the first step in protofibril formation. These results are consistent with recently published photochemical cross-linking data and lend support to recent aggregation mechanisms proposed by Bitan, Teplow, and co-workers [J. Biol. Chem. 2003, 278, 34882-34889].