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)
IsoMS: Automated Processing of LC-MS Data Generated by a Chemical Isotope Labeling Metabolomics Platform
A chemical isotope labeling or isotope
coded derivatization (ICD)
metabolomics platform uses a chemical derivatization method to introduce
a mass tag to all of the metabolites having a common functional group
(e.g., amine), followed by LC-MS analysis of the labeled metabolites.
To apply this platform to metabolomics studies involving quantitative
analysis of different groups of samples, automated data processing
is required. Herein, we report a data processing method based on the
use of a mass spectral feature unique to the chemical labeling approach,
i.e., any differential-isotope-labeled metabolites are detected as
peak pairs with a fixed mass difference in a mass spectrum. A software
tool, IsoMS, has been developed to process the raw data generated
from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair
filtering, and peak-pair intensity ratio calculation. The same peak
pairs detected from multiple samples are then aligned to produce a
CSV file that contains the metabolite information and peak ratios
relative to a control (e.g., a pooled sample). This file can be readily
exported for further data and statistical analysis, which is illustrated
in an example of comparing the metabolomes of human urine samples
collected before and after drinking coffee. To demonstrate that this
method is reliable for data processing, five <sup>13</sup>C<sub>2</sub>-/<sup>12</sup>C<sub>2</sub>-dansyl labeled metabolite standards
were analyzed by LC-MS. IsoMS was able to detect these metabolites
correctly. In addition, in the analysis of a <sup>13</sup>C<sub>2</sub>-/<sup>12</sup>C<sub>2</sub>-dansyl labeled human urine, IsoMS detected
2044 peak pairs, and manual inspection of these peak pairs found 90
false peak pairs, representing a false positive rate of 4.4%. IsoMS
for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS
Development of High-Performance Chemical Isotope Labeling LC–MS for Profiling the Human Fecal Metabolome
Human fecal samples contain endogenous
human metabolites, gut microbiota
metabolites, and other compounds. Profiling the fecal metabolome can
produce metabolic information that may be used not only for disease
biomarker discovery, but also for providing an insight about the relationship
of the gut microbiome and human health. In this work, we report a
chemical isotope labeling liquid chromatography–mass spectrometry
(LC–MS) method for comprehensive and quantitative analysis
of the amine- and phenol-containing metabolites in fecal samples.
Differential <sup>13</sup>C<sub>2</sub>/<sup>12</sup>C<sub>2</sub>-dansyl labeling of the amines and phenols was used to improve LC
separation efficiency and MS detection sensitivity. Water, methanol,
and acetonitrile were examined as an extraction solvent, and a sequential
water–acetonitrile extraction method was found to be optimal.
A step-gradient LC–UV setup and a fast LC–MS method
were evaluated for measuring the total concentration of dansyl labeled
metabolites that could be used for normalizing the sample amounts
of individual samples for quantitative metabolomics. Knowing the total
concentration was also useful for optimizing the sample injection
amount into LC–MS to maximize the number of metabolites detectable
while avoiding sample overloading. For the first time, dansylation
isotope labeling LC–MS was performed in a simple time-of-flight
mass spectrometer, instead of high-end equipment, demonstrating the
feasibility of using a low-cost instrument for chemical isotope labeling
metabolomics. The developed method was applied for profiling the amine/phenol
submetabolome of fecal samples collected from three families. An average
of 1785 peak pairs or putative metabolites were found from a 30 min
LC–MS run. From 243 LC–MS runs of all the fecal samples,
a total of 6200 peak pairs were detected. Among them, 67 could be
positively identified based on the mass and retention time match to
a dansyl standard library, while 581 and 3197 peak pairs could be
putatively identified based on mass match using MyCompoundID against
a Human Metabolome Database and an Evidence-based Metabolome Library,
respectively. This represents the most comprehensive profile of the
amine/phenol submetabolome ever detected in human fecal samples. The
quantitative metabolome profiles of individual samples were shown
to be useful to separate different groups of samples, illustrating
the possibility of using this method for fecal metabolomics studies