9 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
Quantitative Metabolite Profiling Utilizing Parallel Column Analysis for Simultaneous Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Separations Combined with Tandem Mass Spectrometry
In
this work, a fully automated parallel LC column method was established
in order to perform orthogonal hydrophilic interaction chromatography
(HILIC) and reversed-phase (RPLC) chromatography within one analytical
run for targeted quantitative mass spectrometric determination of
metabolites from central carbon metabolism. In this way, the analytical
throughput could be significantly improved compared to previously
established dual separation work flows involving two separate analytical
runs. Two sample aliquots were simultaneously injected onto a dual
column setup columns using a ten-port valve, and parallel separations
were carried out. Sub 2 ÎŒm particle size stationary phases were
employed for both separation methods. HILIC and RPLC eluents were
combined post column followed by ESI-MS/MS detection. The orthogonal
separations were optimized, aiming at an overall separation with 2
retention time segments, while reversed-phase separation was accomplished
within 5.5 min; metabolites on the HILIC phase were retained for a
minimum time of 6 min. The overall run time was 15 min. The setup
was applied to the quantification of 30 primary intercellular metabolites,
including amino acids, organic acids, and nucleotides employing internal
standardization by a fully <sup>13</sup>C-labeled yeast extract. The
comparison with HILICâMS/MS and RPLCâMS/MS in separate
analytical runs revealed that an excellent analytical performance
was achieved by the parallel LC column method. The experimental repeatability
(<i>N</i> = 5) was on average <5% (only for 2 compounds
>5%). Moreover, limits of detection for the new approach ranging
from
0.002â15 ÎŒM were in a good agreement with ones obtained
in separate HILICâMS/MS and RPLCâMS/MS runs (ranging
from 0.01â44 ÎŒM)
Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for <sup>13</sup>CâMetabolic Flux Analysis
For the first time an analytical
work flow based on accurate mass
gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS)
with chemical ionization for analysis providing a comprehensive picture
of <sup>13</sup>C distribution along the primary metabolism is elaborated.
The method provides a powerful new toolbox for <sup>13</sup>C-based
metabolic flux analysis, which is an emerging strategy in metabolic
engineering. In this field, stable isotope tracer experiments based
on, for example, <sup>13</sup>C are central for providing characteristic
patterns of labeled metabolites, which in turn give insights into
the regulation of metabolic pathway kinetics. The new method enables
the analysis of isotopologue fractions of 42 free intracellular metabolites
within biotechnological samples, while tandem mass isotopomer information
is also accessible for a large number of analytes. Hence, the method
outperforms previous approaches in terms of metabolite coverage, while
also providing rich isotopomer information for a significant number
of key metabolites. Moreover, the established work flow includes novel
evaluation routines correcting for isotope interference of naturally
distributed elements, which is crucial following derivatization of
metabolites. Method validation in terms of trueness, precision, and
limits of detection was performed, showing excellent analytical figures
of merit with an overall maximum bias of 5.8%, very high precision
for isotopologue and tandem mass isotopomer fractions representing
>10% of total abundance, and absolute limits of detection in the
femtomole
range. The suitability of the developed method is demonstrated on
a flux experiment of <i>Pichia pastoris</i> employing two
different tracers, i.e., 1,6<sup>13</sup>C<sub>2</sub>-glucose and
uniformly labeled <sup>13</sup>C-glucose
Doping Method Determines Para- or Superparamagnetic Properties of Photostable and Surface-Modifiable Quantum Dots for Multimodal Bioimaging
Semiconductor
quantum dots (QDs) are widely used for optical applications
and bioimaging. In comparison to organic dyes used for fluorescent
labeling, QDs exhibit very high photostability and can be further
surface modified. Equipping QDs with magnetic properties (mQDs) makes
it possible to combine fluorescence and magnetic resonance imaging
analyses. For this purpose, we have prepared water-dispersible and
magnetic CdTe/ZnS mQDs, whereby ferrous ions are selectively incorporated
in either their cores or their shells. This study aims at understanding
the differences in optical, structural, and magnetic properties between
these core- and shell-doped mQDs. Field-dependent isothermal magnetic
susceptibility measurements show that shell-doped mQDs exhibit paramagnetic
and their core-doped equivalents superÂparamagnetic behavior
near room temperature. Shell doping results in about 1.7 times higher
photoluminescence quantum yields and 1.4 times higher doping efficiency
than core doping. X-ray diffraction patterns reveal that core doping
leads to defects in the lattice and hence to a severe decrease in
crystallinity, whereas shell doping has no significant impact on the
crystal structure and consequently fewer disadvantages regarding the
mQDâs quantum yield. These selective doping approaches, particularly
shell doping, allow for the tailored design of paramagnetic QDs having
modifiable and biocompatible particle surfaces. The organic ligandsîžin
this study <i>N</i>-acetyl-l-cysteineîžsufficiently
prevent leakage of toxic metal ions, as shown by cytotoxicity assays
with HepG2 cells. Confocal laser scanning microscopy shows that mQDs
are internalized by these cells and accumulated near their nuclei.
This study shows that biocompatible, fluorescent, and paramagnetic
QDs are promising photostable labels for multimodal bioimaging
An Interlaboratory Evaluation of Drift Tube Ion MobilityâMass Spectrometry Collision Cross Section Measurements
Collision
cross section (CCS) measurements resulting from ion mobilityâmass
spectrometry (IM-MS) experiments provide a promising orthogonal dimension
of structural information in MS-based analytical separations. As with
any molecular identifier, interlaboratory standardization must precede
broad range integration into analytical workflows. In this study,
we present a reference drift tube ion mobility mass spectrometer (DTIM-MS)
where improvements on the measurement accuracy of experimental parameters
influencing IM separations provide standardized drift tube, nitrogen
CCS values (<sup>DT</sup>CCS<sub>N2</sub>) for over 120 unique ion
species with the lowest measurement uncertainty to date. The reproducibility
of these <sup>DT</sup>CCS<sub>N2</sub> values are evaluated across
three additional laboratories on a commercially available DTIM-MS
instrument. The traditional stepped field CCS method performs with
a relative standard deviation (RSD) of 0.29% for all ion species across
the three additional laboratories. The calibrated single field CCS
method, which is compatible with a wide range of chromatographic inlet
systems, performs with an average, absolute bias of 0.54% to the standardized
stepped field <sup>DT</sup>CCS<sub>N2</sub> values on the reference
system. The low RSD and biases observed in this interlaboratory study
illustrate the potential of DTIM-MS for providing a molecular identifier
for a broad range of discovery based analyses
An Interlaboratory Evaluation of Drift Tube Ion MobilityâMass Spectrometry Collision Cross Section Measurements
Collision
cross section (CCS) measurements resulting from ion mobilityâmass
spectrometry (IM-MS) experiments provide a promising orthogonal dimension
of structural information in MS-based analytical separations. As with
any molecular identifier, interlaboratory standardization must precede
broad range integration into analytical workflows. In this study,
we present a reference drift tube ion mobility mass spectrometer (DTIM-MS)
where improvements on the measurement accuracy of experimental parameters
influencing IM separations provide standardized drift tube, nitrogen
CCS values (<sup>DT</sup>CCS<sub>N2</sub>) for over 120 unique ion
species with the lowest measurement uncertainty to date. The reproducibility
of these <sup>DT</sup>CCS<sub>N2</sub> values are evaluated across
three additional laboratories on a commercially available DTIM-MS
instrument. The traditional stepped field CCS method performs with
a relative standard deviation (RSD) of 0.29% for all ion species across
the three additional laboratories. The calibrated single field CCS
method, which is compatible with a wide range of chromatographic inlet
systems, performs with an average, absolute bias of 0.54% to the standardized
stepped field <sup>DT</sup>CCS<sub>N2</sub> values on the reference
system. The low RSD and biases observed in this interlaboratory study
illustrate the potential of DTIM-MS for providing a molecular identifier
for a broad range of discovery based analyses
Additional file 3: of Systems-level organization of yeast methylotrophic lifestyle
Enrichment of the peroxisomal marker protein Pex3p in the peroxisomal fraction. (PDF 271 kb
Additional file 4: of Systems-level organization of yeast methylotrophic lifestyle
Proteomic identification and quantification of methanol metabolic enzymes and control proteins in peroxisomal fractions and homogenates of P. pastoris cells grown on methanol. Containing the following three sheets: Protein hits: contains all identified proteins that met the threshold in at least one sample, with their respective MASCOT scores, number of peptides, and percent sequence coverage. Peptide hits: list of all identified peptides, their MASCOT scores, mass and charge values, and intensities. Peptides used for quantâ+âareas: lists all peptides of the proteins in Table 3 that were used for quantification, and their respective peak areas in the different samples. (XLSX 879 kb
Additional file 1: of Systems-level organization of yeast methylotrophic lifestyle
Transcriptomic, proteomic, and metabolomic regulation of P. pastoris during methylotrophic growth. Containing the following eight sheets: Summary Omics Data: number of significantly regulated genes, proteins or metabolites (e.g. âupâ refers to up-regulation in methanol/glycerol compared to glucose). Transcriptomics and proteomics: Average fold changes and P values of transcriptomics and proteomics comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat. Average values derive from three biological replicates per condition. Metabolomics: Average fold changes and P values of metabolomics measurements comparing P. pastoris cultivated with methanol/glycerol or glucose as carbon source in chemostat cultivations. Average values derive from three biological replicates per condition. Co-regulation (related to Fig. 1 in the text): Regulation of the 575 gene-protein pairs with transcriptomics and proteomics data available and assignment to regulatory groups. Central carbon metabolism (related to Fig. 4 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 4. Amino acid metabolism (related to Fig. 6 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 6. Vitamin biosynthesis (related to Fig. 7 in the text): Average fold changes and P values of transcriptomics, proteomics, and metabolomics measurement depicted in Fig. 7. Peroxisomal gene regulation: Average fold changes and P values of transcriptomics and proteomics for all mentioned peroxisomal genes. Average values derive from three biological replicates per condition. (XLSX 2348 kb