Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites

Article abstract of DOI:10.1002/anie.202107101

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Full text of DOI:10.1002/anie.202107101

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Chemoselective and highly sensitive quantification of gut
microbiome and human metabolites
Authors: Weifeng Lin, Louis P. Conway, Miroslav Vujasinovic, J.-
Matthias Löhr, and Daniel Globisch
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202107101
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10.1002/anie.202107101
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RESEARCH ARTICLE
Chemoselective and highly sensitive quantification of gut
microbiome and human metabolites
Weifeng Lin, Louis P. Conway, Miroslav Vujasinovic, J.-Matthias Löhr, and Daniel Globisch*
Abstract: The microbiome has a fundamental impact on the human
host’s physiology through the production of highly reactive
compounds that can lead to disease development. One class of such
compounds are carbonyl-containing metabolites, which are involved
in diverse biochemical processes. Mass spectrometry is the method
of choice for analysis of metabolites but carbonyls are analytically
challenging. Herein, we have developed a new chemical biology tool
using chemoselective modification to overcome analytical limitations.
Two isotopic probes allow for the simultaneous and semi-quantitative
analysis at femtomole as well as qualitative analysis at attomole
quantities that allows for detection of more than 200 metabolites in
human fecal, urine and plasma samples. This comprehensive mass
spectrometric analysis enhances the scope of metabolomics-driven
biomarker discovery. We anticipate that our Chemical Biology tool will
be of general use in metabolomics analysis to obtain a better
understanding of microbial interactions with the human host and
disease development.
their role in human physiology, techniques for the selective
discovery and analysis of these metabolites are urgently required
so that their bioactivity and impact on the human host can be
better studied.[4]
Carbonyl-containing metabolites are
a highly reactive
compound class that can form conjugates with DNA and proteins.
They are also involved in a wide range of biochemical pathways
and their dysregulation has been linked to a host of pathological
conditions.[5] Furthermore, six carbonyl-containing metabolites
have been classified as carcinogens by the International Agency
for Research of Cancer.[6] Reactive oxygen species (ROS) are
one important part of carbonyl metabolites that play a crucial role
in cell signaling in many biochemical processes. However, the
deregulation of ROS can lead to reaction of carbonyl species with
cellular macromolecules resulting in the unfavorable disruption of
normal physiology.[7] The pathologies of diseases such as cancer,
retinopathy and asthma have been associated with the imbalance
between the ROS production and antioxidant defenses.[8]
Deciphering the interactions of reactive microbial
metabolites with their host is therefore of critical importance.
Liquid chromatography-coupled mass spectrometry (LC-MS) is
ideally suited for discovery of biomarkers and new metabolites in
complex biological matrices due to its sensitivity, dynamic range,
and resolution.[9] However, many carbonyl-containing metabolites
possess poor ionization properties making them exceedingly
difficult to analyze.[10] It is crucial to develop selective and
sensitive analytical methods to obtain a greater overview of this
reactive compound class in biological systems. Derivatization
methods have been developed for carbonyl-containing
metabolites to improve their analysis and stability.[7b, 11] However,
those methods are limited due to mass spectrometric
interferences by complex matrices and to analysis of a low
number of compounds only. We have recently developed a
chemoselective probe immobilized to magnetic beads for the
capture of metabolites in human samples.[12] This method enables
increased mass spectrometric sensitivity by up to a factor of one
million due to efficient removal of the unreacted matrix
background through magnetic separation and improved ionization
properties of the captured and tagged metabolites after release
from the immobilized beads.
Introduction
Trillions of microbes colonize the surfaces and cavities of
the human body and particularly the gastrointestinal tract.[1] These
communities are heavily metabolically active and exchange
metabolites with other microbes and their host.[2] One of the
beneficial relationships of these co-evolved microbial
communities with their host is protection against pathogenic
infections. In contrast, abnormal alterations of the composition of
the gut microbiota, termed microbiota dysbiosis, has been linked
to disease development and other pathologies.[3] While
metagenomic sequencing has revealed up- and downregulated
bacterial species, the molecular mechanisms underpinning
communication between the host and the microbiota are largely
unknown.[1c] The microbiota genetic pool contains about 400
times more genetic information than the human genome and
many of these genes encode metabolic reactions orthogonal to
the human metabolism that constitute
xenobiotics.[2c,
bioactivity and more likely remain to be discovered. In order to
understand the importance of microbiota-derived metabolites and
a major source of
Many of these compounds are of unknown
2d]
Here, we report the second generation of these probes,
which have been redesigned to enable a new chemical biology
methodology for advanced mass spectrometric analysis that we
[a]
M.Sc. Weifeng Lin, Dr. L. P. Conway, and Prof. Dr. D. Globisch
Department of Chemistry - BMC, Science for Life Laboratory
Uppsala University, Box 599, SE-75124 Uppsala, Sweden
E-mail: Daniel.globisch@scilifelab.uu.se
have
termed
Quantitative
Sensitive
CHEmoselective
MetAbolomics (quant-SCHEMA). The synthesis of 13C/12C
isotopically labeled analogues of the probe and separate sample
treatment allows for comparative and quantitative analysis of
endogenous and microbiota-derived metabolites in human
samples at low concentrations. While isotope-labeling is a well-
established part of the (chemo)proteomic workflow, it is rarely
used in metabolomics due to the higher structural diversity of
metabolites and concentration differences.[13] The few reports are
based on simple chemistry without separation from the sample
matrix, require harsh conditions for coupling, are limited in the
[b]
[c]
*
Dr. Miroslav Vujasinovic, Prof. Dr. J.-Matthias Löhr
Department for Digestive Diseases, Karolinska University Hospital,
Stockholm, Sweden
Prof. Dr. J.-Matthias Löhr
Department of Clinical Science, Intervention and Technology
(CLINTEC), Karolinska Institute, Stockholm, Sweden
To whom correspondence should be addressed
Supporting information for this article is given via a link at the end
of the document.
1
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Figure 1. Chemoselective probe for quantitative analysis of metabolites in human samples.a) Chemical structure of chemoselective probe (1 and 13C6-
1) immobilized to magnetic beads.  = amino-activated magnetic beads;  = bioorthogonal cleavage site;  = metabolite tag containing isotope-labeled moiety;
 = reactive site. b) Schematic workflow for analysis of human urine, plasma and fecal samples. After metabolite extraction, the metabolome of two different
samples is treated with either ‘light’ (1) or ‘heavy’ beads (13C6-1). After separation of captured metabolites, bioorthogonal cleavage of both combined bead types
releases tagged metabolites for UPLC-MS analysis. c) Chemical synthesis of chemoselective probe and immobilization to magnetic beads. Reagents and
conditions: i) benzoyl chloride 1.2 equiv., Et3N 3.0 equiv., DCM, room temperature (r.t.) 3 h. ii) Pd/H2, MeOH, r.t., 5 h. iii) N,N-Diisopropylethylamine (DIPEA)
3.0 equiv., DCM, r.t., 5 h. iv) Trifluoroacetic acid (TFA)/ DCM 1:1, r.t., 2 h. v) 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
1.2 equiv., hydroxybenzotriazole (HOBT) 1.1 equiv., DIPEA 3.0 equiv., DCM, r.t., 16 h. vi) 2M aqueous LiOH, MeOH/H2O 1:1, r.t., 30 min. vii) PyBop 1.5 equiv.,
HOBT 1.1 equiv., DIPEA 3.0 equiv., DMF/DCM 1:1, r.t., 16 h.
validation of metabolites, or have only been applied to a single
Results and Discussion
14]
sample type.[11a,
This second generation of chemoselective
probes described in this study now facilitates quantitative analysis
that is an important improvement for comparative analysis of two
sample sets. The redesign of the probe scaffold that only contains
the bioorthogonal cleavage site from the first-generation probe
and now provides several advantages: i) quantitative and
comparative analysis between two sample sets by use of a light
and a heavy labeled probe; ii) a new coupling chemistry to amino-
activated magnetic beads facilitates a shorter synthetic route; iii)
the reactive site is introduced to a primary amine for more efficient
activation; iv) precise quantification of selected metabolites at low
fmol concentrations without the need of commercially available
isotope labeled compounds; v) treatment of three different human
sample types with the identical procedure; and vi) biomarker
discovery using a combination of isotope-labeled chemoselective
probe methodology and bioinformatic metabolomics analysis.
Elimination of undesired mass spectrometric matrix
interferences and this new ionizable-tag technology leads to
substantially enhanced detection sensitivity of metabolites at
attomole quantities. This method allows for the parallel
quantitative analysis of known metabolites and yet unidentified
metabolite. This is important for biomarker discovery as only an
estimated 2% of metabolites has been detected in the human
metabolome.[15] Analysis of carbonyls in human fecal, plasma and
urine samples collected from pancreatic cancer patients led to
discovery of a series of unknown metabolites of this reactive
compound class as well as a previously inaccessible overview of
carbonyl regulation in humans.
This new quantitative methodology is based on
chemoselective probe 1 that is immobilized to magnetic beads
and a corresponding stable isotopically labelled analogue 13C6-1
(Figure 1a). The availability of two analogues that only differ in
their mass but not in chemophysical properties allows for direct
comparative mass spectrometric analysis of two sample sets.
Human plasma and urine samples collected at two different time
points were treated with the unlabeled (time point 1) and labeled
(time point 2) chemoselective probe (Figure 1b). Fecal samples
were treated with the same methodology to investigate
microbiome-derived metabolites. After separate incubation for 16
hours to capture carbonyl-containing metabolites, the beads were
isolated from each sample using a magnet, washed, and
combined for bioorthogonal cleavage. The resulting tagged
metabolites of the general structure 2/13C6-2 were analyzed using
ultra performance liquid chromatography coupled to mass
spectrometry (UPLC-MS). The obtained data was analyzed using
the bioinformatic software package XCMS in R.[16] Both
conjugated metabolites 2 and 13C6-2 can be distinguished by their
mass difference of 6.0201 Da without interference from the
natural isotope distribution, while both elute at the same retention
time due to the same physicochemical properties. These
properties increase the accuracy and precision of the analysis and
allow for a direct readout of relative changes between each
captured metabolite in the two samples. The straightforward
synthesis begins with benzoylation of the secondary amine in
Boc- and Cbz-protected diethylenetriamine 3 using either natural
or 13C6-labeled benzoic acid (Figure 1c). After Cbz-deprotection
of intermediate 4, the free amine was coupled to the p-
2
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RESEARCH ARTICLE
alkylaldehyde, 2,3-pentanedione as an alkylketone and
phenylacetaldehyde as an aromatic aldehyde that is sterically
hindered. Comparison of an aqueous solution of these
compounds (c = 100 µM each) to human urine samples with
metabolites spiked in at the same concentration resulted in a
recovery rate of 49-67% with an average RSD of 8.2% (Table S2).
Our method was designed for straightforward identification of the
captured carbonyl-containing metabolites via bioinformatic
analysis using XCMS through the difference of  = 6.0201 Da in
the MS chromatogram (Figure 2a). The efficiency and
reproducibility were tested using a pooled fecal sample from ten
patients to maximize the number of carbonyl-containing
metabolites. The sample was split in two equal parts and each
was either treated with the light (1) or the heavy 13C-labeled
chemoselective probe (13C6-1), the conjugates were cleaved from
the magnetic beads after magnetic separation and analyzed using
UPLC-MS analysis. For the bioinformatic analysis, we used
unmodified beads as a control sample that were treated with the
same cleavage conditions to resemble the same mass
spectrometric background. This experiment led to the
identification of 239 metabolite pairs according to these criteria: i)
difference of 6.0201 Da; ii) within retention time < 0.05 min; iii)
mass larger than the free probe (281.1608 Da); and iv)
abundance significantly larger than the control sample. This
number is greater than identified in fecal samples in any other
study and exceeds our first-generation analysis by a factor of
two.[9c, 12a] The reproducibility of this multistep method is illustrated
by plotting the ratios of 13C/12C carbonyl-conjugates, which is
commonly used for determination of the reproducibility in related
chemoproteomics research (Figure 2b). This similarity of labeled
and unlabeled probes is required for precise sample analysis as
it accounts for differences between both synthetic compounds
1/13C6-1, coupling efficiency to the magnetic beads, metabolite
conjugation, bioorthogonal cleavage, and UPLC-MS analysis.
This analysis was followed by an additional reproducibility
test in urine samples from one patient but collected 6 months
apart. Samples T1 and T2 were split in three parts each and each
aliquot of T1 was treated with probe 1, while each aliquot of T2
was treated with probe 13C6-1. In this reproducibility experiment,
60 out of 82 pairs of labeled and unlabeled tagged metabolites
were detected in all independently analyzed samples (81% of
metabolite pairs), which were identified out of 3,127 common
features (Figure 2c). These 60 metabolite pairs were found to be
up- and down-regulated in the same direction in repeated
analyses, which is demonstrated by the T2/T1 ratios with relative
standard deviations of up- and down-regulated features of 21.4%
and 17.9%, respectively (Figures 2d and S3).
Figure 2. Method validation. a) Treatment of pooled fecal sample by both
heavy and light chemoselective probe that can be separately analyzed in
UPLC-MS experiments with  = 6.0201 Da with the example of conjugated
acetaldehyde 2a/13C6-2a. b) 13C-probe/12C-probe ratios for 239 identified
pairs in pooled fecal samples demonstrates the similarity of both
treatments. The mean and SD are highlighted in green. A complete list of
validated metabolites and m/z values for these carbonyl-metabolites is
reported in Figure S5 and Table S4. c) Reproducibility experiment of the
same urine samples collected from one patient at two different time points.
Venn diagram illustrates common global features and identified metabolite
pairs among three independent experiments. d) Detailed illustration of
measured ratios from three independent experiments in urine samples.
Data are presented as mean ± SD from independent experimental triplicate
(N = 3).
nitrocinnamyloxycarbonyl (Noc)-moiety 5 that serves as the
bioorthogonal cleavage site.[12c] In a two-step synthetic procedure,
intermediate
6 was functionalized with the Boc-protected
alkoxyamine to yield the full-length probe 7. This intermediate was
saponified to obtain 8 for coupling to amino-activated magnetic
beads under standard peptide-coupling conditions and TFA
treatment furnishes chemoselective probe 1.
Maximizing the coverage of carbonyls during the mass
spectrometric analysis requires the capture of metabolites with
high selectivity, optimized ionization properties and solubility of
the released tagged metabolite. Conjugates of acetone (2b) and
butanone (2c) were synthesized from common precursor 10 in a
straightforward three-step synthetic route (Figure 3a). These
purified conjugates were utilized for LOD and LOQ experiments.
The acetone-conjugate 2b can be detected at a concentration of
100 pM, which corresponds to an LOD of 500 amol. Furthermore,
the LOQ for conjugates of acetone (2b) and butanone (2c) using
this new method is c = 0.5-5 nM. To also include the reaction
efficiency, we have also performed LOD measurements with
varying concentrations of the carbonyl-metabolite and a constant
concentration of the 13C-labeled reagent. The LOD values for
We have conducted a series of experiments to validate this
new method for metabolite analysis in biological samples. We
initially determined the bead load capacity for the coupling of the
chemoselective probe that was applied for all following
experiments at 0.8 ± 0.1 µmol/g (Figure S1). As the next step, we
determined the capture efficiency to be an average of 51.3% for
five selected metabolites (acetaldehyde, acetone, butanone,
valeraldehyde, and dihydroxyacetone / Table S1). An additional
analysis of equimolar solutions for each metabolite as well as 1:3
and 3:1 ratios demonstrates the quality of this methodology
(Figure S2). We have also determined the recovery rate for three
selected carbonyl compounds that are representative for different
reactivities of carbonyls in human samples: octanal as an
3
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RESEARCH ARTICLE
Figure 3. Improved mass spectrometric properties and structure validation. a) Synthetic scheme of conjugated acetone 2b and conjugated butanone 2c
as well as their corresponding LOD/LOQ experiments. Limit of detection (LOD) was determined by signal to noise ratio > 3, and limit of quantification (LOQ)
was determined by signal to noise ratio > 10. Reagent and condition: i) TFA/ DCM 1:1, r.t., 2 h, ii) HBTU 1.2 equiv., HOBT 1.1 equiv., DIPEA 3.0 equiv., DCM,
r.t., 16 h. iii) acetone or butanone as solvent, r.t., 16 h. b) Workflow for the metabolite validation using conjugate standard metabolites with 2-octanone 2d as
an example. c) List of metabolites according to their sources and with association to diseases.
acetone and butanone were in a similar range and for seven
additional carbonyls were determined to be between 500 amol -
500 fmol (Table S5).
development and can now be investigated in parallel (Figure
3c).[18]
As fecal samples contain an abundance of microbiota-
derived metabolites generated in the human gut, this sample type
represents a readout for microbiome metabolism. Acetaldehyde
and acetone are volatile compounds that are common metabolic
endpoints of many biochemical pathways in the microbiome.[19]
Other examples include pyruvic acid and 4-hydroxyphenylpyruvic
acid that are excreted from E. coli but originate from different
Based on the reproducibility of the relative quantitative
method and the highly increased mass spectrometric sensitivity,
we constructed a chemical library of unlabeled metabolite
conjugates of the general structure 2 (Figure 3b). These reference
compounds are required for structure validation of the captured
carbonyl conjugates through comparison of retention times and
co-injection MS experiments (Figure S4 and Table S3).
Additionally, the structural E/Z isomers of non-symmetric
aldehydes and ketones after reaction with the alkoxyamine
reagent can readily be identified and analyzed in parallel.[12a, 17]
Structure validation is critical as it is the bottleneck of
metabolomics analysis and is key to biochemical analysis.[15] We
chemically synthesized 107 biologically relevant conjugates 2
using the same straightforward synthetic route as for the LOD
experiments for direct UPLC-MS experiments.
metabolic processes. 5-Keto-D-gluconic acid is
a known
metabolite from the Gluconobacter genus, and is produced from
D-gluconate metabolism through catalysis by D-gluconate
dehydrogenase (GADH).[20] These compounds have been linked
to the development of diverse diseases and demonstrate the
ability of our procedure to track changes in concentrations of
biologically-relevant molecules in three of the most common
sample types.
Human urine and plasma samples are the most commonly
investigated sample types for metabolomics-driven biomarker
discovery as these ‘liquid biopsies’ are readily accessible.[21]
While plasma samples represent a snapshot of ongoing metabolic
pathways, urine samples contain the metabolic endproducts of
Using this comprehensive library, we validated 60
metabolites in human fecal samples (Figure S5). These
metabolites were identified in at least one of the samples and
ranged from volatile metabolites acetone or acetaldehyde to
dietary compounds, e.g. furfural or kaempferol. To our knowledge, the human clearance process as well as xenobiotics including
this number of validated metabolites has not been exceeded by
any previous study, and nine of these metabolites have been
detected for the first time in this sample type.[11a, 11b] Carbonyl
isomers co-eluted in three cases and could not be distinguished
from each other despite the use of standards. We thus analyzed
these regioisomers combined. A series of captured metabolites,
which were either of microbial origin, food products or
endogenous metabolites, have previously been linked to disease
microbiome-derived compounds. For this study, we collected
urine and plasma samples from the same high-risk patients for
pancreatic cancer at two different time points T1 and T2. We have
applied our developed quant-SCHEMA method for direct
comparative analysis of both time points for each of the three
individuals. The total number of detected carbonyls in these
samples, confirmed by the pairs of the light and heavy tags, are
180 metabolites for plasma and 203 for urine samples (N = 6;
4
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Figure 4. Large-scale analysis in human patient samples. a) Venn diagrams of the peak pair numbers detected in the plasma (N=6) and urine samples
(N=6). b) Principal component analysis (PCA) of plasma and urine samples for three individuals at different time points. c) Heatmap of the top 50 common
carbonyl-pairs in the plasma and urine analysis (p-value, ANOVA). d) Time-dependent fold-change of validated carbonyl-metabolites in plasma and urine
samples. The complete data output from this experiment is shown as a heatmap in Figure S6. e) Investigated metabolites in the sorbitol metabolism pathway in
plasma and urine samples.
Figure 4a). Our analysis revealed distinct pattern of metabolites
in all investigated patient samples with 119 and 98 metabolites
common to all three investigated plasma and urine samples,
respectively. This reliable extraction and isolation of large
numbers of carbonyl metabolites enables the large-scale analysis
of this reactive compound class in human samples for biomarker
discovery and is enhanced to previous studies due to the
increased MS sensitivity and metabolite identification procedure.
The treated urine and plasma samples were analyzed in parallel
with the same control sample in a randomized sequence allowing
for comparison of the samples through principal components
analysis (Figure 4b). This unsupervised multivariate analysis
clearly separated plasma and urine samples, but also clearly
grouped samples according to collection time, while replicates
remained tightly clustered.
As samples were collected from the same individuals, the
carbonyl-composition can be directly compared in both sample
types (Figures 4c and S6). Our mass spectrometric investigation
into carbonyl-metabolites revealed distinct individual pattern
within the 50 most significant carbonyl-conjugates that were
detected in all six samples. This analysis revealed distinct
signatures for individuals as well as collection timepoints.
5
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Interestingly, the five metabolites butanal, acetaldehyde, acetone,
valeraldehyde, and diacetone alcohol follow the same changes in
plasma and urine over time, which are considered volatile organic
compounds (VOCs). VOCs are endproducts of different
biochemical pathways or can be produced by pathogenic
microorganisms.[22] Importantly, some of these compounds have
been proposed as potential biomarkers for early-stage diagnosis
of diverse diseases. However, due to their volatile properties, this
compound class is difficult to detect using conventional LC-MS
analysis. Our isotope-labeling-based methodology of this reactive
compound class facilitates measurement over time in urine and
plasma samples that is a first step towards monitoring of these
important metabolites in humans. By taking a closer look at
changes of metabolites with validated structures, we identified
individual differences (Figure 4d). We observed four metabolites
upregulated in both sample types for each of the patients at the
second
timepoint
(hydroxyacetone,
glycolaldehyde,
acetylacetone, and acetoin). It is also intriguing that acetaldehyde
and butanal were different for each individual but has the same
tendency in urine and plasma samples. This provides the
opportunity to monitor changes of these metabolically important
metabolites over time in both sample types in parallel.
Furthermore, the xenobiotic cyclopentanone was detected in two
urine samples, while cyclohexanone was solely detected in the
samples from one patient (P10 and U10). Notably, the five
microbial metabolites acetaldehyde, acetone, pyruvic acid, 4-
hydroxyphenylpyruvic acid, and 5-keto-D-gluconic acid present in
fecal samples were also detected in plasma and urine samples.
Important to note that in the latter two sample types, these
metabolites can either be products from the host or microbiome
metabolism.
The broad applicability of the method has been
demonstrated through the investigation of three different human
sample types. The analysis of human plasma and urine samples
of high-risk patients at two different timepoints allows for robust
mapping and monitoring of metabolic changes in high-risk
patients. Importantly, our method permits identification of
metabolic variations in individuals over time that can in the future
be utilized to identify disease onset. This has so far been
unavailable for broad coverage of carbonyl-metabolites in clinical
human samples. While this method has been developed for
metabolite profiling of the highly reactive carbonyl compound
class, it can also be extended to analysis of other functionalities
through modification of the reactive site  (Figure 1a).
Figure 5. Quantification of acetone. a) Workflow for quantification of acetone
in urine and fecal samples. b) Calibration curve of conjugated acetone (2b). c),
Quantification results of acetone in three urine samples at different time points.
Data are presented as mean ± SD from experimental triplicates (N = 3). ns: not
significant, p > 0.05; *: p < 0.05; **: p <0.01. d) Comparison of quantification
and semi-quantitative metabolomics results. e), Quantification results of
acetone in a fecal sample from one individual. Data are presented as mean ±
SD from experimental quadruplicates (N = 4).
responses such as statins treatment and breast cancer
neoadjuvant chemotherapy.[24] In the present study, the precise
carbonyl-containing metabolite profiling of this longitudinal
sample cohort has elucidated specific individual metabolic
changes that can now be monitored in larger cohorts to identify
signatures of disease onset and progression (Figure S6). The
current clinical diagnosis and medication of diseases is generally
applicable for the majority of the human population. However,
symptoms relapse and unexpected side effects are inevitable for
some patients owing to the genomic and metabolic differences of
individuals. Personalized or precision medicine has been
proposed in the past decades for disease management by taking
individual’s genomic variations, biochemical profiling and lifestyle
into account for decisions on disease treatment.[25] Our method
with the advantageous sensitivity for mass spectrometric
investigation of this reactive compound class has a high potential
for profiling this carbonyl-metabotyping of individuals towards
personalized diagnostics and medicine after future validation in
large cohorts.
Quant-SCHEMA additionally allows for mapping and
quantification of a wide range of metabolic pathways. Examples
are the sorbitol and ascorbic acid metabolic pathways that contain
carbonyl metabolites. Both are major pathways for endogenous
production of glyoxal through autoxidation of glycolaldehyde
(Figure 4e and S7). Glyoxal is an α-oxoaldehyde that is produced
by different organisms including bacteria and humans by glucose
oxidation, lipid peroxidation, and DNA oxidation. Additionally, this
metabolite is a typical member of the highly reactive 1,2-
dicarbonyl compounds that has been investigated due to its
detrimental effect on the gut microbiota.[23] The significantly up-
regulated values of glycolaldehyde in all individuals in both
plasma and urine samples might be an indication for increased
oxidative damage in these individuals, but requires further
investigation.
As our quant-SCHEMA analysis is based on isotope-labeled
metabolite tags, it allows not only for the identification of
metabolites but also their precise quantification in complex
biological matrices. Additionally, our method provides a new
straightforward possibility to synthesize 13C6-labelled standards
for the precise quantification of a metabolite of interest. This is
advantageous as not all metabolites are commercially available
as stable isotope labelled analogues. We sought to quantify
acetone as an important example to further validate our results
from the large-scale carbonyl screening in Figure 4d. Acetone is
a volatile metabolite and difficult to quantify using LC-MS analysis.
It is produced by different metabolic pathways as well as the gut
microbiome with importance in diseases such as diabetes.[27] We
synthesized light and heavy labelled standards of the acetone
conjugate 2b and 13C6-2b and used these to measure a
calibration curve enabling quantification of acetone in urine and
fecal samples (Figures 5a and 5b).[9a, 28] The three urine samples
Metabolomics analyses are being more commonly applied
for investigation and prediction of the individual treatment
6
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Figure 6. Metabolite discovery. a) Overview of identified metabolites in all three investigated sample types based on different levels of confidence. b) 21
newly discovered metabolites in each sample type. c) Representative metabolite detection procedure for conjugated 2-undecanone (2e) and acetylacetone
(2f) in urine, plasma and fecal samples compared to the synthetic standard.
were treated with the unlabeled probe 1 and the isotope labeled
acetone-standard (13C6-2b) was spiked into the sample before the
bioorthogonal cleavage. The quantification experiments were
highly reproducible and identified significant changes in acetone
concentration in urine samples from two individuals (Figure 5c,
any reported study (Figure 3c). Five carbonyl metabolites in urine,
13 in plasma and nine in fecal samples were detected for the first
time, which belong to diverse compound classes (Figure 6b). It is
important to note that some of these 27 metabolites have
previously been detected in another sample type.[18] Our
evaluation process to determine the presence or absence of
metabolites in the investigated three different sample types, is
depicted in Figure 6c for the conjugates of 2-undecanone (2e) and
acetylacetone (2f). The correct conjugate 2e was identified next
to an unknown regioisomer and was only detected in plasma
samples, while 2f was only present in plasma and urine samples.
average RSD
=
14.5%). Importantly, the quantification
experiments perfectly mirrored our metabolomics-based readout
from the comparative screening for urine samples (Figures 4d and
5d). This method was also applied for quantification of acetone in
fecal samples, which has never been performed before. We
determined the quantity of acetone to be 0.97 ± 0.16 fmol / mg
feces in four independent sample preparation and treatment
experiments from the same individual. This demonstrates the high
sensitivity and reproducibility of this mass spectrometric method
and that the loss of volatile compounds during sample preparation
is negligible (Figure 5e).
Our sensitive mass spectrometric analysis also led to the
identification of previously undetected metabolites in all three
investigated sample types. Due to removal of the captured
metabolites from the complex sample matrix using magnetic
separation, analysis in parallel of metabolites present in all three
human sample types is possible. The retention time and peak
shape for our analysis is identical for every sample type. This is
an important advantage as the magnetic separation removes the
common matrix effect that other derivatization methods and
standard metabolomics studies need to consider through
additional normalization procedures.[26] Validation of carbonyl-
containing metabolites with authentic conjugate standards led to
43 and 42 identified metabolites in urine and plasma, respectively
(Figure 6a and S8). With 60 validated metabolites in fecal
samples, this is the highest numbers of validated metabolites in
Conclusion
In summary, the developed method represents a new and
reproducible chemical biology tool that enhances metabolomics
and metabolite analysis in complex human samples. This new
methodology is based on a chemoselective probe immobilized to
magnetic beads with a light and heavy isotope tag that allows for
advanced analysis compared to other derivatization methods for
metabolite analysis, which lack separation from the sample matrix
and must overcome matrix interferences. Due to the combination
of natural and isotope-labeled conjugates before LC-MS analysis,
this new method does not require any normalization procedure
including internal standards or normalization of different sample
types. This is necessary in general metabolomics analysis to
reduce technical errors. The parallel and quantitative analysis of
metabolites at attomole quantities and detection of more than 200
metabolites in human fecal, urine and plasma samples has not
been reported before. This method also facilitates detection of 21
7
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10.1002/anie.202107101
Angewandte Chemie International Edition
RESEARCH ARTICLE
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Acknowledgements
We are grateful for funding by the Swedish Research Council (VR
2016-04423 / VR 2020-04707), the Swedish Cancer Foundation
(19 0347 Pj), Carl Tryggers Foundation (CTS 2018:820), and a
generous start-up grant from the Science for Life Laboratory to
D.G. This study made use of the NMR Uppsala infrastructure,
which is funded by the Department of Chemistry - BMC and the
Disciplinary Domain of Medicine and Pharmacy.
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Keywords: Bioorganic chemistry • chemoselectivity • mass
spectrometry • metabolites • microbiome
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RESEARCH ARTICLE
Entry for the Table of Contents
Chemoselective probes based on stable isotope labeling allow for highly sensitive mass
spectrometric quantification and detection of metabolites at attomole quantities in three
human sample types.
9
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