Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. 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 (K0) 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

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 Ų). Solutions favoring the <i>A</i> state yielded several well-defined families of significantly unfolded conformations (1800–2000 Ų) matching in size conformations between the <i>A</i> 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 <i>A</i> state, however, is charged more extensively than the native state during ESI and decays more rapidly following ESI. <i>A</i> state ions unfold on a time scale equal to or shorter than the experiment (≤50 ms) to more extended structures

Diphenylalanine Self Assembly: Novel Ion Mobility Methods Showing the Essential Role of Water

The mechanism and driving forces behind the formation of diphenylalanine (FF) nanotubes have attracted much attention in the past decades. The hollow structure of the nanotubes suggests a role for water during the self-assembly process. Here, we use novel ion-mobility mass spectrometry methods to probe the early oligomers formed by diphenylalanine peptides. Interestingly, water-bound oligomers are observed in nano-electrospray ionization (ESI) mass spectra in the absence of bulk solvent. In addition, ligated water clusters transit the ion mobility cell but (often) dissociate before detection. These water molecules are shown to be essential for the formation of diphenylalanine oligomers larger than the dimer. The ligated water molecules exist in the solvent free environment either as neutral water or as protonated water clusters, depending on the composition of solvent from which they are sprayed. Water adduction helps stabilize conformers that are otherwise energetically unstable ultimately leading to the assembly of FF nanotubes

Dimerization of Chirally Mutated Enkephalin Neurotransmitters: Implications for Peptide and Protein Aggregation Mechanisms

We have probed the structures and aggregation propensities of chirally substituted [Ala²]-Leu-Enkephalin peptides (i.e., Leu-Enkephalin G2A) with a combination of ion-mobility spectrometry/mass spectrometry and techniques of computational chemistry. Our IMS/MS data reveal a strong correlation between the propensity to form peptide dimers and the subsequent aggregation propensity. This correlation indicates that the dimerization process is fundamental to the overall self-assembly process. Our computational data correlate a conformational conversion during the peptide association process with a reduced experimental dimer formation and subsequent aggregation propensity. Furthermore, our analysis indicates that monomer activation does not precede peptide association and thus suggests that the entire-refolding or gain-in-interaction models are more realistic accounts of the peptide self-assembly process than the monomer-conversion model. In sum, our results suggest that conformational transitions of early peptide oligomers represent bottlenecks of the peptide self-assembly process and thus highlight the importance of structurally characterizing this reaction during amyloid formation

Phenylalanine Oligomers and Fibrils: The Mechanism of Assembly and the Importance of Tetramers and Counterions

Phenylalanine is the only amino acid known to self-assemble into toxic fibrillar aggregates. An elevated concentration of phenylalanine in the blood can result in Phenylketonuria, a progressive mental retardation. Ion-mobility mass spectrometry is employed to investigate the structure and distribution of phenylalanine oligomers formed in the early stage of the aggregation cascade. The experimental cross sections indicate that phenyl-alanine self-assembles at neutral pH into oligomers composed of multiple layers of four monomers. The monomers arrange themselves to create a hydrophilic core made of zwitterionic termini and expose hydrophobic aromatic side chains to the outside. At high pH, the interactions between the neutral amino and negatively charged carboxylate of phenylalanine allow a minor population of ladder-like oligomers to be formed and detected in ion-mobility experiments. However, counterions such as ammonium rearrange those structures into the same structures observed at neutral pH. The cytotoxicity of Phe oligomers and fibrils may be due to favorable interactions between the hydrophobic exterior and the cell membrane and strong interactions between the hydrophilic core of Phe oligomers and ions, resulting in ion leakage and cellular damage

Amyloid β‑Protein Assembly: Differential Effects of the Protective A2T Mutation and Recessive A2V Familial Alzheimer’s Disease Mutation

Oligomeric states of the amyloid β-protein (Aβ) appear to be causally related to Alzheimer’s disease (AD). Recently, two familial mutations in the amyloid precursor protein gene have been described, both resulting in amino acid substitutions at Ala2 (A2) within Aβ. An A2V mutation causes autosomal recessive early onset AD. Interestingly, heterozygotes enjoy some protection against development of the disease. An A2T substitution protects against AD and age-related cognitive decline in non-AD patients. Here, we use ion mobility-mass spectrometry (IM-MS) to examine the effects of these mutations on Aβ assembly. These studies reveal different assembly pathways for early oligomer formation for each peptide. A2T Aβ42 formed dimers, tetramers, and hexamers, but dodecamer formation was inhibited. In contrast, no significant effects on Aβ40 assembly were observed. A2V Aβ42 also formed dimers, tetramers, and hexamers, but it did not form dodecamers. However, A2V Aβ42 formed trimers, unlike A2T or wild-type (wt) Aβ42. In addition, the A2V substitution caused Aβ40 to oligomerize similar to that of wt Aβ42, as evidenced by the formation of dimers, tetramers, hexamers, and dodecamers. In contrast, wt Aβ40 formed only dimers and tetramers. These results provide a basis for understanding how these two mutations lead to, or protect against, AD. They also suggest that the Aβ N-terminus, in addition to the oft discussed central hydrophobic cluster and C-terminus, can play a key role in controlling disease susceptibility

Familial Alzheimer’s Disease Mutations Differentially Alter Amyloid β-Protein Oligomerization

Although most cases of Alzheimer’s disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer’s disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide’s assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD

Amyloid β‑Protein Assembly: The Effect of Molecular Tweezers CLR01 and CLR03

The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer’s disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment

Interactions between Amyloid‑β and Tau Fragments Promote Aberrant Aggregates: Implications for Amyloid Toxicity

We have investigated at the oligomeric level interactions between Aβ(25–35) and Tau(273–284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer’s disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau­(273–284) shows a high affinity to form heteroligomers with existing Aβ(25–35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25–35) or Tau(273–284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25–35) fibrils. The incorporation of some Tau into β-rich Aβ(25–35) oligomers reduces the aggregation propensity of Aβ(25–35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25–35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves

Aggregation of Chameleon Peptides: Implications of α‑Helicity in Fibril Formation

We investigate the relationship between the inherent secondary structure and aggregation propensity of peptides containing chameleon sequences (i.e., sequences that can adopt either α or β structure depending on context) using a combination of replica exchange molecular dynamics simulations, ion-mobility mass spectrometry, circular dichroism, and transmission electron microscopy. We focus on an eight-residue long chameleon sequence that can adopt an α-helical structure in the context of the iron-binding protein from <i>Bacillus anthracis</i> (PDB id 1JIG) and a β-strand in the context of the baculovirus P35 protein (PDB id 1P35). We show that the isolated chameleon sequence is intrinsically disordered, interconverting between α-helical and β-rich conformations. The inherent conformational plasticity of the sequence can be constrained by addition of flanking residues with a given secondary structure propensity. Intriguingly, we show that the chameleon sequence with helical flanking residues aggregates rapidly into fibrils, whereas the chameleon sequence with flanking residues that favor β-conformations has weak aggregation propensity. This work sheds new insights into the possible role of α-helical intermediates in fibril formation