7 research outputs found
Recommendations for reporting ion mobility mass spectrometry measurements
Š 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. Š 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc
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 âatypicalâ
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
Dimerization of Chirally Mutated Enkephalin Neurotransmitters: Implications for Peptide and Protein Aggregation Mechanisms
We have probed the structures and aggregation propensities
of chirally
substituted [Ala<sup>2</sup>]-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
Differentiation of CC vs CXC Chemokine Dimers with GAG Octasaccharide Binding Partners: An Ion Mobility Mass Spectrometry Approach
Chemokines,
8 kDa proteins implicated in leukocyte migration via
oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation
response as a means to regulate chemokine migration. Structural characterization
of chemokines non-covalently bound to GAGs provides physiologically
meaningful data in regard to routine inmmunosurveillance and disease
response. In order to analyze the structures resulting from the GAG:chemokine
interaction, we employed ion mobility mass spectrometry (IMMS) to
analyze monocyte chemoattractant proteinâ1 (MCPâ1),
a CC chemokine, and interleukinâ8 (ILâ8), a CXC chemokine,
along with their individual interactions with GAG heparin octasaccharides.
We show that MCPâ1 and ILâ8 are physiologically present
as a dimer, with MCPâ1 having two variants of its dimeric form
and ILâ8 having only one. We also show that the MCPâ1
dimer adopts two conformations, one extended and one compact, when
bound to a dodecasulfated heparin octasaccharide. Binding of MCPâ1
to heparin octasaccharide isomers of varying sulfation patterns results
in similar arrival time distribution values, which suggests minimal
distinguishing features among the resultant complexes. Additionally,
tandem mass spectrometry (MS/MS) showed that the binding of MCPâ1
to a heparin octasaccharide has different dissociation patterns when
compared with the corresponding ILâ8 bound dimer. Overall,
IMMS and MS/MS were used to better define the structural tendencies
and differences associated with CC and CXC dimers when associated
with GAG octasaccharides
A Transferable, Sample-Independent Calibration Procedure for Trapped Ion Mobility Spectrometry (TIMS)
Ion mobility spectrometry-mass
spectrometry (IMS-MS) determines
momentum transfer cross sections of ions to elucidate their structures.
Recent IMS methods employ electrodynamic fields or nonstationary buffer
gases to separate ions. These methods require a calibration procedure
to determine ion mobilities from the experimental data. This applies
in particular to trapped IMS (TIMS), a novel IMS method with reported
high resolving powers. Here, we report the first systematic assessment
of the accuracy and the limitations of mobility calibration in TIMS.
Our data show that the currently used TIMS calibration approach reproduces
drift tube mobilities to approximately 1% (95th percentile). Furthermore,
we develop a transferable and sample-independent calibration procedure
for TIMS. The central aspects of our approach are (1) a calibration
function derived from a solution to the Boltzmann transport equation
and (2) calibration constants based on a Taylor expansion of instrument
properties (TEIP). The key advantage of our calibration approach over
current ones is its transferability: one equation and one set of parameters
are sufficient to calibrate ion mobilities for various instrument
settings, compound classes, or charge states. Our approach is transferable
over time and sufficiently accurate (âź1â2%) for structure-elucidation
purposes. While we develop our calibration procedure specifically
for TIMS, the approach we take is generic in nature and can be applied
to other IMS systems
Ion Mobility Spectrometry Reveals the Mechanism of Amyloid Formation of Aβ(25â35) and Its Modulation by Inhibitors at the Molecular Level: Epigallocatechin Gallate and <i>Scyllo</i>-inositol
Amyloid cascades leading 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 the cellular machinery. The relationship between the transformation
from natively unstructured assembly to the β-sheet oligomers
to disease is important in understanding disease onset and the development
of therapeutic agents. Research on this early oligomeric region has
largely been unsuccessful since traditional techniques measure only
ensemble average oligomer properties. Here, ion mobility methods are
utilized to deduce the modulation of peptide self-assembly pathways
in the amyloid-β protein fragment Aβ(25â35) by
two amyloid inhibitors (epigallocatechin gallate and <i>scyllo</i>-inositol) that are currently in clinical trials for Alzheimerâs
Disease. We provide evidence that suppression of β-extended
oligomers from the onset of the conversion into β-oligomer conformations
is essential for effective attenuation of β-structured amyloid
oligomeric species often associated with oligomer toxicity. Furthermore,
we demonstrate the ease with which ion mobility spectrometryâmass
spectrometry can guide the development of therapeutic agents and drug
evaluation by providing molecular level insight into the amyloid formation
process and its modulation by small molecule assembly modulators
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