10 research outputs found
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 ÎĽm 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 HILIC–MS/MS and RPLC–MS/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
0.002–15 μM were in a good agreement with ones obtained
in separate HILIC–MS/MS and RPLC–MS/MS runs (ranging
from 0.01–44 μM)
Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics
In
this work, simultaneous targeted metabolic profiling by isotope
dilution and non-targeted fingerprinting is proposed for cancer cell
studies. The novel streamlined metabolomics workflow was established
using anion-exchange chromatography (IC) coupled to high-resolution
mass spectrometry (MS). The separation time of strong anion-exchange
(2 mm column, flow rate 380 μL min<sup>–1</sup>, injection
volume 5 ÎĽL) could be decreased to 25 min for a target list
comprising organic acids, sugars, sugar phosphates, and nucleotides.
Internal standardization by fully <sup>13</sup>C labeled <i>Pichia
pastoris</i> extracts enabled absolute quantification of the
primary metabolites in adherent cancer cell models. Limits of detection
(LODs) in the low nanomolar range and excellent intermediate precisions
of the isotopologue ratios (on average <5%, <i>N</i> = 5, over 40 h) were observed. As a result of internal standardization,
linear dynamic ranges over 4 orders of magnitude (5 nM–50 μM, <i>R</i><sup>2</sup> > 0.99) were obtained. Experiments on drug-sensitive
versus resistant SW480 cancer cells showed the feasibility of merging
analytical tasks into one analytical run. Comparing fingerprinting
with and without internal standard proved that the presence of the <sup>13</sup>C labeled yeast extract required for absolute quantification
was not detrimental to non-targeted data evaluation. Several interesting
metabolites were discovered by accurate mass and comparing MS2 spectra
(acquired in ddMS2 mode) with spectral libraries. Significant differences
revealed distinct metabolic phenotypes of drug-sensitive and resistant
SW480 cells
Integrated Exposomics/Metabolomics for Rapid Exposure and Effect Analyses
The totality of environmental
exposures and lifestyle factors,
commonly referred to as the exposome, is poorly understood. Measuring
the myriad of chemicals that humans are exposed to is immensely challenging,
and identifying disrupted metabolic pathways is even more complex.
Here, we present a novel technological approach for the comprehensive,
rapid, and integrated analysis of the endogenous human metabolome
and the chemical exposome. By combining reverse-phase and hydrophilic
interaction liquid chromatography (HILIC) and fast polarity-switching,
molecules with highly diverse chemical structures can be analyzed
in 15 min with a single analytical run as both column’s effluents
are combined before analysis. Standard reference materials and authentic
standards were evaluated to critically benchmark performance. Highly
sensitive median limits of detection (LODs) with 0.04 ÎĽM for
>140 quantitatively assessed endogenous metabolites and 0.08 ng/mL
for the >100 model xenobiotics and human estrogens in solvent were
obtained. In matrix, the median LOD values were higher with 0.7 ng/mL
(urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the
dual-column approach’s applicability, real-life urine samples
from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure
scenario) were assessed in a targeted and nontargeted manner. Our
liquid chromatography high-resolution mass spectrometry (LC-HRMS)
approach demonstrates the feasibility of quantitatively and simultaneously
assessing the endogenous metabolome and the chemical exposome for
the high-throughput measurement of environmental drivers of diseases
Integrated Exposomics/Metabolomics for Rapid Exposure and Effect Analyses
The totality of environmental
exposures and lifestyle factors,
commonly referred to as the exposome, is poorly understood. Measuring
the myriad of chemicals that humans are exposed to is immensely challenging,
and identifying disrupted metabolic pathways is even more complex.
Here, we present a novel technological approach for the comprehensive,
rapid, and integrated analysis of the endogenous human metabolome
and the chemical exposome. By combining reverse-phase and hydrophilic
interaction liquid chromatography (HILIC) and fast polarity-switching,
molecules with highly diverse chemical structures can be analyzed
in 15 min with a single analytical run as both column’s effluents
are combined before analysis. Standard reference materials and authentic
standards were evaluated to critically benchmark performance. Highly
sensitive median limits of detection (LODs) with 0.04 ÎĽM for
>140 quantitatively assessed endogenous metabolites and 0.08 ng/mL
for the >100 model xenobiotics and human estrogens in solvent were
obtained. In matrix, the median LOD values were higher with 0.7 ng/mL
(urine) and 0.5 ng/mL (plasma) for exogenous chemicals. To prove the
dual-column approach’s applicability, real-life urine samples
from sub-Saharan Africa (high-exposure scenario) and Europe (low-exposure
scenario) were assessed in a targeted and nontargeted manner. Our
liquid chromatography high-resolution mass spectrometry (LC-HRMS)
approach demonstrates the feasibility of quantitatively and simultaneously
assessing the endogenous metabolome and the chemical exposome for
the high-throughput measurement of environmental drivers of diseases
Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for <sup>13</sup>C‑Metabolic 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
Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS
Laser ablation (LA) in combination with inductively coupled
plasma
time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of
elements from the entire mass range for every pixel, regardless of
the isotopes of interest for a certain application. This provides
nontargeted multi-element (bio-)imaging capabilities and the unique
possibility to screen for elements that were initially not expected
in the sample. Quantification of a large range of elements is limited
as the preparation of highly multiplexed calibration standards for
bioimaging applications by LA-ICP-(TOF)MS is challenging. In this
study, we have developed a workflow for semiquantitative analysis
by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards.
The presented approach is intended for the mapping of biological samples
due to the requirement of matrix-matched standards for accurate quantification
in LA-ICPMS, a prerequisite that is given by the use of gelatin-based
standards. A library of response factors was constructed based on
72 elements for the semiquantitative calculations. The presented method
was evaluated in two stages: (i) on gelatin samples with known elemental
concentrations and (ii) on real-world samples that included prime
examples of bioimaging (mouse spleen and tumor tissue). The developed
semiquantification approach was based on 10 elements as calibration
standards and provided the determination of 136 nuclides of 63 elements,
with errors below 25%, and for half of the nuclides, below 10%. A
web application for quantification and semiquantification of LA-ICP(-TOF)MS
data was developed, and a detailed description is presented to easily
allow others to use the presented method
A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies
Lipid identification
and quantification are essential objectives
in comprehensive lipidomics studies challenged by the high number
of lipids, their chemical diversity, and their dynamic range. In this
work, we developed a tailored method for profiling and quantification
combining (1) isotope dilution, (2) enhanced isomer separation by
C30 fused-core reversed-phase material, and (3) parallel Orbitrap
and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer.
The combination of parallelizable ion analysis without time loss together
with different fragmentation techniques (HCD/CID) and an inclusion
list led to higher quality in lipid identifications exemplified in
human plasma and yeast samples. Moreover, we used lipidome isotope-labeling
of yeast (LILY)î—¸a fast and efficient in vivo labeling strategy
in <i>Pichia pastoris</i>î—¸to produce (nonradioactive)
isotopically labeled eukaryotic lipid standards in yeast. We integrated
the <sup>13</sup>C lipids in the LC-MS workflow to enable relative
and absolute compound-specific quantification in yeast and human plasma
samples by isotope dilution. Label-free and compound-specific quantification
was validated by comparison against a recent international interlaboratory
study on human plasma SRM 1950. In this way, we were able to prove
that LILY enabled quantification leads to accurate results, even in
complex matrices. Excellent analytical figures of merit with enhanced
trueness, precision and linearity over 4–5 orders of magnitude
were observed applying compound-specific quantification with <sup>13</sup>C-labeled lipids. We strongly believe that lipidomics studies
will benefit from incorporating isotope dilution and LC-MSn strategies
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 quant + areas: 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. “up” 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