Recommendations for reporting ion mobility Mass Spectrometry measurements

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

Protomers of Benzocaine: Solvent and Permittivity Dependence

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid, which finds an application as a local anesthetic (LA), is found to adopt in its protonated form at least two populations of distinct structures in the gas phase and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization (ESI) process. Here we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry (IM-MS) to yield gas-phase IR spectra of simultaneously m/z and drift-time resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species - the N- and O-protonated form. Density functional theory (DFT) calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation has been suggested

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

Factors That Drive Peptide Assembly and Fibril Formation: Experimental and Theoretical Analysis of Sup35 NNQQNY Mutants

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single β-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early β-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms

The Solution Assembly of Biological Molecules Using Ion Mobility Methods: From Amino Acids to Amyloid β-Protein

Ion mobility spectrometry-mass spectrometry (IMS-MS) methods are increasingly used to study noncovalent assemblies of peptides and proteins. This review focuses on the noncovalent self-assembly of amino acids and peptides, systems at the heart of the amyloid process that play a central role in a number of devastating diseases. Three different systems are discussed in detail: the 42-residue peptide amyloid-β42 implicated in the etiology of Alzheimer's disease, several amyloid-forming peptides with 6-11 residues, and the assembly of individual amino acids. We also discuss from a more fundamental perspective the processes that determine how quickly proteins and their assemblies denature when the analyte ion has been stripped of its solvent in an IMS-MS measurement and how to soften the measurement so that biologically meaningful data can be recorded

Elucidating Structures of Protein Complexes by Collision-Induced Dissociation at Elevated Gas Pressures

Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes. We show that CID of the homotetrameric protein complexes streptavidin (53 kDa), neutravidin (60 kDa), and concanavalin A (110 kDa) provides access to all subunits of the investigated protein complexes, including structurally informative dimers. We report on an “atypical” dissociation pathway, which for concanavalin A proceeds via symmetric partitioning of the precursor charges and produces dimers with the same charge states that were previously reported from surface induced dissociation. Our data suggest a correlation between the formation of subunits by CID in tandem-TIMS/MS, their binding strengths in the native tetramer structures, and the applied activation voltage. Ion mobility spectra of in situ-generated subunits reveal a marked structural heterogeneity inconsistent with annealing into their most stable gas phase structures. Structural transitions are observed for in situ-generated subunits that resemble the transitions reported from collision-induced unfolding of natively folded proteins. These observations indicate that some aspects of the native precursor structure is preserved in the subunits generated from disassembly of the precursor complex. We rationalize our observations by an approximately 100-fold shorter activation time scale in comparison to traditional CID in a collision cell. Finally, the approach discussed here to conduct CID at elevated pressures appears generally applicable also for other types of tandem-ion mobility spectrometers

Metastability of Protein Solution Structures in the Absence of Solvent: Rugged Energy Landscape and Glass-Like Behavior

Despite the significance of differentially modified proteins (proteoforms) to human health, it remains challenging to identify how proteoforms alter protein structural dynamics and function. Although native ion mobility/mass spectrometry is well-suited to handle proteoform heterogeneity, it characterizes protein structures in the absence of solvent. This raises long-standing, unanswered questions about the biological significance of structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the structural denaturation of the protein ubiquitin in the solvent-free environment. We show that ubiquitin exists in the absence of solvent as an ensemble of kinetically stable subpopulations that are separated by substantial free energy barriers. These subpopulations unfold but do not interconvert, which indicates that the solvent-free subpopulations originate from different solution-phase conformations. The subpopulations exhibit stretched-exponential denaturation kinetics consistent with a glass transition associated with separating the C-terminal beta-strand from the N-terminal beta-hairpin occurring on the folded side of the unfolding transition state. Our data indicate that this transition state is highly polarized with significant native content in the N-terminal beta-hairpin and alpha-helix, resembling the transition state reported for the presence of a solvent. Taken together, our analysis suggests that ubiquitin in the solvent-free environment reflects the conformational ensemble of ubiquitin in solution because the initially formed solvent-free state of ubiquitin appears glass-like and “melts” over several seconds

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