5 research outputs found
5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry
We
describe a new set of isotope reagents, <sup>12</sup>C<sub>4</sub>-, <sup>12</sup>C<sub>2</sub><sup>13</sup>C<sub>2</sub>-, and <sup>13</sup>C<sub>4</sub>-5-diethylamino-naphthalene-1-sulfonyl chloride
(DensCl), in combination with liquid chromatography Fourier-transform
ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved
analysis of the amine- and phenol-containing submetabolome. The synthesis
of the reagents is reported, and an optimized derivatization protocol
for labeling amines and phenols is described. To demonstrate the utility
of the triplex reagents for metabolome profiling of biological samples,
urine samples collected daily from a healthy volunteer over a period
of 14 days were analyzed. The overall workflow is straightforward,
including differential isotope labeling of individual samples and
a pooled sample that serves a global internal standard, mixing of
the isotope differentially labeled samples and LC-MS analysis for
relative metabolome quantification. Comparing to the dansyl chloride
(DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer
the advantages of improved metabolite detectability due to enhanced
sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling
vs about 600 peak pairs detected by DnsCl labeling) and analysis speed
(i.e., simultaneous analysis of two comparative samples by DensCl
vs only one comparative sample analyzed by DnsCl)
5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry
We
describe a new set of isotope reagents, <sup>12</sup>C<sub>4</sub>-, <sup>12</sup>C<sub>2</sub><sup>13</sup>C<sub>2</sub>-, and <sup>13</sup>C<sub>4</sub>-5-diethylamino-naphthalene-1-sulfonyl chloride
(DensCl), in combination with liquid chromatography Fourier-transform
ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved
analysis of the amine- and phenol-containing submetabolome. The synthesis
of the reagents is reported, and an optimized derivatization protocol
for labeling amines and phenols is described. To demonstrate the utility
of the triplex reagents for metabolome profiling of biological samples,
urine samples collected daily from a healthy volunteer over a period
of 14 days were analyzed. The overall workflow is straightforward,
including differential isotope labeling of individual samples and
a pooled sample that serves a global internal standard, mixing of
the isotope differentially labeled samples and LC-MS analysis for
relative metabolome quantification. Comparing to the dansyl chloride
(DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer
the advantages of improved metabolite detectability due to enhanced
sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling
vs about 600 peak pairs detected by DnsCl labeling) and analysis speed
(i.e., simultaneous analysis of two comparative samples by DensCl
vs only one comparative sample analyzed by DnsCl)
5‑Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry
We
describe a new set of isotope reagents, <sup>12</sup>C<sub>4</sub>-, <sup>12</sup>C<sub>2</sub><sup>13</sup>C<sub>2</sub>-, and <sup>13</sup>C<sub>4</sub>-5-diethylamino-naphthalene-1-sulfonyl chloride
(DensCl), in combination with liquid chromatography Fourier-transform
ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved
analysis of the amine- and phenol-containing submetabolome. The synthesis
of the reagents is reported, and an optimized derivatization protocol
for labeling amines and phenols is described. To demonstrate the utility
of the triplex reagents for metabolome profiling of biological samples,
urine samples collected daily from a healthy volunteer over a period
of 14 days were analyzed. The overall workflow is straightforward,
including differential isotope labeling of individual samples and
a pooled sample that serves a global internal standard, mixing of
the isotope differentially labeled samples and LC-MS analysis for
relative metabolome quantification. Comparing to the dansyl chloride
(DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer
the advantages of improved metabolite detectability due to enhanced
sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling
vs about 600 peak pairs detected by DnsCl labeling) and analysis speed
(i.e., simultaneous analysis of two comparative samples by DensCl
vs only one comparative sample analyzed by DnsCl)
Development of High-Performance Chemical Isotope Labeling LC–MS for Profiling the Carbonyl Submetabolome
Metabolites
containing a carbonyl group represent several important
classes of molecules including various forms of ketones and aldehydes
such as steroids and sugars. We report a high-performance chemical
isotope labeling (CIL) LC–MS method for profiling the carbonyl
submetabolome with high coverage and high accuracy and precision of
relative quantification. This method is based on the use of dansylhydrazine
(DnsHz) labeling of carbonyl metabolites to change their chemical
and physical properties to such an extent that the labeled metabolites
can be efficiently separated by reversed phase LC and ionized by electrospray
ionization MS. In the analysis of six standards representing different
carbonyl classes, acetaldehyde could be ionized only after labeling
and MS signals were significantly increased for other 5 standards
with an enhancement factor ranging from ∼15-fold for androsterone
to ∼940-fold for 2-butanone. Differential <sup>12</sup>C- and <sup>13</sup>C-DnsHz labeling was developed for quantifying metabolic
differences in comparative samples where individual samples were separately
labeled with <sup>12</sup>C-labeling and spiked with a <sup>13</sup>C-labeled pooled sample, followed by LC–MS analysis, peak
pair picking, and peak intensity ratio measurement. In the replicate
analysis of a 1:1 <sup>12</sup>C-/<sup>13</sup>C-labeled human urine
mixture (<i>n</i> = 6), an average of 2030 ± 39 pairs
per run were detected with 1737 pairs in common, indicating the possibility
of detecting a large number of carbonyl metabolites as well as high
reproducibility of peak pair detection. The average RSD of the peak
pair ratios was 7.6%, and 95.6% of the pairs had a RSD value of less
than 20%, demonstrating high precision for peak ratio measurement.
In addition, the ratios of most peak pairs were close to the expected
value of 1.0 (e.g., 95.5% of them had ratios of between 0.67 and 1.5),
showing the high accuracy of the method. For metabolite identification,
a library of DnsHz-labeled standards was constructed, including 78
carbonyl metabolites with each containing MS, retention time (RT),
and MS/MS information. This library and an online search program for
labeled carbonyl metabolite identification based on MS, RT, and MS/MS
matches have been implemented in a freely available Website, www.mycompoundid.org. Using
this library, out of the 1737 peak pairs detected in urine, 33 metabolites
were positively identified. In addition, 1333 peak pairs could be
matched to the metabolome databases with most of them belonging to
the carbonyl metabolites. These results show that <sup>12</sup>C-/<sup>13</sup>C-DnsHz labeling LC–MS is a useful tool for profiling
the carbonyl submetabolome of complex samples with high coverage
Development of Isotope Labeling Liquid Chromatography Mass Spectrometry for Mouse Urine Metabolomics: Quantitative Metabolomic Study of Transgenic Mice Related to Alzheimer’s Disease
Because of a limited volume of urine
that can be collected from
a mouse, it is very difficult to apply the common strategy of using
multiple analytical techniques to analyze the metabolites to increase
the metabolome coverage for mouse urine metabolomics. We report an
enabling method based on differential isotope labeling liquid chromatography
mass spectrometry (LC–MS) for relative quantification of over
950 putative metabolites using 20 μL of urine as the starting
material. The workflow involves aliquoting 10 μL of an individual
urine sample for <sup>12</sup>C-dansylation labeling that target amines
and phenols. Another 10 μL of aliquot was taken from each sample
to generate a pooled sample that was subjected to <sup>13</sup>C-dansylation
labeling. The <sup>12</sup>C-labeled individual sample was mixed with
an equal volume of the <sup>13</sup>C-labeled pooled sample. The mixture
was then analyzed by LC–MS to generate information on metabolite
concentration differences among different individual samples. The
interday repeatability for the LC–MS runs was assessed, and
the median relative standard deviation over 4 days was 5.0%. This
workflow was then applied to a metabolomic biomarker discovery study
using urine samples obtained from the TgCRND8 mouse model of early
onset familial Alzheimer’s disease (FAD) throughout the course
of their pathological deposition of beta amyloid (Aβ). It was
showed that there was a distinct metabolomic separation between the
AD prone mice and the wild type (control) group. As early as 15–17
weeks of age (presymptomatic), metabolomic differences were observed
between the two groups, and after the age of 25 weeks the metabolomic
alterations became more pronounced. The metabolomic changes at different
ages corroborated well with the phenotype changes in this transgenic
mice model. Several useful candidate biomarkers including methionine,
desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic
acid were identified. Some of them were found in previous metabolomics
studies in human cerebrospinal fluid or blood samples. This work illustrates
the utility of this isotope labeling LC–MS method for biomarker
discovery using mouse urine metabolomics