6 research outputs found
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
Online Deuterium Hydrogen Exchange and Protein Digestion Coupled with Ion Mobility Spectrometry and Tandem Mass Spectrometry
Online deuterium hydrogen exchange
(DHX) and pepsin digestion (PD)
is demonstrated using drift tube ion mobility spectrometry (DTIMS)
coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron
transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin,
followed by subsequent quenching and digestion, is performed within
∼60 s, yielding 100% peptide sequence coverage. The high reproducibility
of the IMS separation allows spectral feature matching between two-dimensional
IMS-MS datasets (undeuterated and deuterated) without the need for
dataset alignment. Extracted ion drift time distributions (XIDTDs)
of deuterated peptic peptides are mobility-matched to corresponding
XIDTDs of undeuterated peptic peptides that were identified using
collision-induced dissociation (CID). Matching XIDTDs allows a straightforward
identification and deuterium retention evaluation for labeled peptides.
Aside from the mobility separation, the ion trapping capabilities
of the LTQ, combined with ETD, are demonstrated to provide single-residue
resolution. Deuterium retention for the <i>c</i>- series
ions across residues M<sup>1</sup>–L<sup>15</sup> and N<sup>25</sup>–R<sup>42</sup> are in good agreement with the known
secondary structural elements within ubiquitin
Ion Mobility, Hydrogen/Deuterium Exchange, and Isotope Scrambling: Tools to Aid Compound Identification in ‘Omics Mixtures
Liquid
chromatography tandem mass spectrometry (LC-MS/MS), a widely
used method for comparative ‘omics analysis, experiences challenges
with compound identification due to matrix effects, difficulty in
separating isomer and isobaric ions, and long analysis times. Ion
mobility spectrometry (IMS) has proven to be useful in separating
isomer and isobar ions; however, IMS-MS suffers from decreased peak
capacity due to the correlation in ion size and mass. In proof-of-principle
experiments, the use of gas-phase hydrogen/deuterium exchange (HDX)
combined with IMS-MS/MS techniques is demonstrated to offer advantages
for compound identification. Measurements providing unique information
for ions include <i>m</i>/<i>z</i> value, drift
time in He buffer gas, drift time in He and D<sub>2</sub>O buffer
gases, deuterium incorporation pattern (isotopic distribution), deuterium
incorporation pattern after collisional activation, and fragment ion
deuterium incorporation pattern upon collision-induced dissociation
(CID). These techniques are here shown to be highly reproducible (drift
time coefficients of variation < 1.0% and isotopic pattern root-mean-square
deviations of 0.5–1.5%) while demonstrating an increased ability
to distinguish individual molecules from diverse classes of compounds
(peptides, catecholamines, nucleosides, amino acids, etc.). The concept
of using such (and similar) information for rapid, high-throughput
molecular identification via database searching of standard libraries
is briefly discussed, and an example of such usage is presented for
a bonafide metabolite extract sample
Conformation Types of Ubiquitin [M+8H]<sup>8+</sup> Ions from Water:Methanol Solutions: Evidence for the N and A States in Aqueous Solution
Ion mobility and mass spectrometry measurements are used
to examine
the gas-phase populations of [M+8H]<sup>8+</sup> ubiquitin ions formed
upon electrospraying 20 different solutions from 100:0 to 5:95 water:methanol
that are maintained at pH = 2.0. Over this range of solution conditions,
mobility distributions for the +8 charge state show substantial variations.
Here we develop a model that treats the combined measurements as one
data set. By varying the relative abundances of a discrete set of
conformation types, it is possible to represent distributions obtained
from any solution. For solutions that favor the well-known A-state
ubiquitin, it is possible to represent the gas-phase distributions
with seven conformation types. Aqueous conditions that favor the native
structure require four more structural types to represent the distribution.
This analysis provides the first direct evidence for trace amounts
of the A state under native conditions. The method of analysis presented
here should help illuminate how solution populations evolve into new
gas-phase structures as solvent is removed. Evidence for trace quantities
of previously unknown states under native solution conditions may
provide insight about the relationship of dynamics to protein function
as well as misfolding and aggregation phenomena
Huntingtin N‑Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues
Early
stage oligomer formation of the huntingtin protein may be
driven by self-association of the 17-residue amphipathic α-helix
at the protein’s N-terminus (Nt17). Oligomeric structures have
been implicated in neuronal toxicity and may represent important neurotoxic
species in Huntington’s disease. Therefore, a residue-specific
structural characterization of Nt17 is crucial to understanding and
potentially inhibiting oligomer formation. Native electrospray ion
mobility spectrometry–mass spectrometry (IMS-MS) techniques
and molecular dynamics simulations (MDS) have been applied to study
coexisting monomer and multimer conformations of Nt17, independent
of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer
ion structures comprise a helix-turn-coil configuration and a helix-extended-coil
region. Elongated dimer species comprise partially helical monomers
arranged in an antiparallel geometry. This stacked helical bundle
may represent the earliest stages of Nt17-driven oligomer formation.
Nt17 monomers and multimers have been further probed using diethylpyrocarbonate
(DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6
are modified at higher DEPC concentrations, which led to the formation
of an intermediate monomer structure. These modifications resulted
in decreased extended monomer ion conformers, as well as a reduction
in multimer formation. From the MDS experiments for the dimer ions,
Lys6 residues in both monomer constituents interact with Ser16 and
Glu12 residues on adjacent peptides; therefore, the decrease in multimer
formation could result from disruption of these or similar interactions.
This work provides a structurally selective model from which to study
Nt17 self-association and provides critical insight toward Nt17 multimerization
and, possibly, the early stages of huntingtin exon 1 aggregation
A New Ion Mobility–Linear Ion Trap Instrument for Complex Mixture Analysis
A new
instrument that couples a low-pressure drift tube with a
linear ion trap mass spectrometer is demonstrated for complex mixture
analysis. The combination of the low-pressure separation with the
ion trapping capabilities provides several benefits for complex mixture
analysis. These include high sensitivity, unique ion fragmentation
capabilities, and high reproducibility. Even though the gas-phase
separation and the mass measurement steps are each conducted in an
ion filtering mode, detection limits for mobility-selected peptide
ions are in the tens of attomole range. In addition to ion separation,
the low-pressure drift tube can be used as an ion fragmentation cell
yielding mobility-resolved fragment ions that can be subsequently
analyzed by multistage tandem mass spectrometry (MS<sup>n</sup>) methods
in the ion trap. Because of the ion trap configuration, these methods
can be comprised of any number (limited by ion signal) of collision-induced
dissociation (CID) and electron transfer dissociation (ETD) processes.
The high reproducibility of the gas-phase separation allows for comparison
of two-dimensional ion mobility spectrometry (IMS)–MS data
sets in a pixel-by-pixel fashion without the need for data set alignment.
These advantages are presented in model analyses representing mixtures
encountered in proteomics and metabolomics experiments