New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites

Article abstract of DOI:10.1039/c8sc01502c

Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

Full text of DOI:10.1039/c8sc01502c

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and D. Globisch, Chem. Sci., 2018, DOI: 10.1039/C8SC01502C.
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New enzymatic and mass spectrometric methodology for the
selective investigation of gut microbiota-derived metabolites
Caroline Ballet,‡a Mário S. P. Correia,‡a Louis P. Conway,a Theresa L. Locher,a Laura C. Lehmann,b
Neeraj Garg,a Miroslav Vujasinovic,c Sebastian Deindl,b J.-Matthias Löhr,cd and Daniel Globisch*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-
metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of
metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new
enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human
urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in
the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown
and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass
spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with
state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-
derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other
metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.
www.rsc.org/
dysregulated gut microflora significantly contributes to a
variety of diseases including cancer, diabetes, obesity,
cardiovascular diseases, and inflammatory bowel disease
Introduction
One of the most exciting scientific developments in the
past decade has been the understanding that gut microbiota
profoundly impact human physiology.1-3 This complex
consortium of trillions of microbes performs a diverse range of
biotransformation reactions orthogonal to the host’s own
metabolic pathways. The human microbiota has even been
referred to as an additional human organ as it influences
crucial pathways, including nutrition, detoxification,
metabolism, hormonal homeostasis, immune tolerance, and
especially inflammation. Mounting evidence indicates that a
(IBD).4-6 These microbial communities are highly metabolically
active and constantly exchange metabolites with other
microbes and their human host. Our knowledge of the overall
metabolic interactions of microbial communities with their
host is still limited. Detailed elucidation of this metabolic
interaction is required to identify unknown metabolites and to
evaluate their toxic or beneficial properties on the human
host.7, 8
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Scheme 1
Workflow of the targeted metabolomics-driven identification and mass spectrometric analysis of sulfated metabolites including the chemical structure of four
representative gut microbiota-derived metabolites 1-4 identified in human samples.
To allow for a detailed metabolic investigation we sought
to develop a selective method for the discovery of unknown
Sulfatase assay
Each sulfated compound was dissolved in 50 mM ammonium
acetate at a concentration of 0.2 mM and treated either with
130 U of crude arylsulfatase mixture or with 19 U of purified
arylsulfatase. The reaction mixture was incubated and shaken
for 24 h (300 rpm, 21 °C) and the enzyme precipitated by
addition of cold methanol (4 × the assay volume). The samples
were vigorously shaken for 30 s and cooled for 15 min at 0 °C.
The supernatant was removed after centrifugation (13780 g
for 5 min) and dried in vacuo. The remaining pellet was
dissolved in 60 µl water/acetonitrile (95/5, v/v), vigorously
shaken for 30 s and transferred to HPLC-vials for UPLC-MS/MS
analysis.
metabolites
derived
from
the
co-metabolism
of
microorganisms and their host. It has been reported that gut
microbiota profoundly affect phase I and phase II conversions,
which have evolved to clear xenobiotic metabolites in
mammals.9 Phase I metabolism enzymes such as cytochrome
P450 oxidases introduce polar groups into xenobiotics
including microbiota derived metabolites. This process is
followed by phase II conjugation to enhance hydrophilicity.
This mechanism has evolved to excrete these metabolites from
the human body, however, it can also lead to bioactivation and
the generation of toxic compounds. In particular, O-sulfated
metabolites have been identified as a major feature of the co-
metabolism of microbes and their host.10 Metabolites in this
class have been suggested to be key regulators of bacterial
interaction with their host as sulfotransferases and sulfatases
have been identified in the genomes of gut microbes.11, 12 Two
Urine sample preparation
Ice cold methanol (4 mL) was added to urine sample aliquots
(1 mL) for protein precipitation. Each sample was vigorously
shaken for 30 s and then cooled at 4 °C for 30 min.
prominent examples are indoxyl sulfate (
1) and p-cresyl sulfate
(
2
), which were among the first identified metabolites of
Fecal sample preparation
microbial and host co-metabolism (Scheme 1).12 Indole and
cresol are produced from the aromatic amino acids,
tryptophan and tyrosine, through digestion by gut microbes.
These molecules are absorbed in the colon of the human host
and further metabolized by phase I and II clearance processes.
Both metabolites have since been described as uremic toxins
as they are linked to the development of chronic kidney
disease (CKD) as well as cardiovascular diseases, emphasizing
Frozen and homogenized fecal samples were cut, lyophilized
overnight and suspended in water/DMSO solution (95/5, v/v; 1
mL/60 mg dry fecal sample). Afterwards, the suspension was
centrifuged at 4 °C for 5 min (13780 g) and the supernatant
was collected. This step was followed by protein precipitation
with the addition of ice cold methanol (4 × volume of the fecal
extract). Each sample was vigorously shaken for 30 s and
cooled at -20 °C for 30 min.
the relevance of this metabolite class to human physiology.13,
14
General metabolite extraction and sample work-up
The discovery of unknown metabolic links between
microbes and their human host is crucial to evaluate their
impact on disease development as well as to identify new
biomarkers or drug targets. Analytical tools for the selective
analysis of microbial metabolites are not available. To
overcome this limitation, we have developed a sulfatase assay
for the facile and selective hydrolysis of sulfate esters under
mild conditions (Scheme 1). This approach is in line with
examples from proteome (proteases) and modified nucleic
acid analyses (phosphatases) and represents a new strategy
for targeted metabolomics analysis.
Upon protein precipitation and centrifugation at 13780 g for
5 min, the supernatant containing the extracted fecal or urine
metabolite mixture was dried in vacuo at ambient
temperature. The residue was dissolved in 400 µL of 50 mM
ammonium acetate buffer (300 µL for feces) and divided into
two equal parts for enzymatic and control assays. An aliquot of
the purified arylsulfatase was utilized in the enzymatic assay
(11 U for urine / 38 U for feces), while the volume of the
control assay was adjusted to the same volume with 50 mM
ammonium acetate buffer. An aliquot of arylsulfatase solution
was denatured by heating to 99 °C for 5 min and used as
negative control in the enzymatic assay. Both assays were
shaken (300 rpm) for 17 h at 21 °C and subjected to protein
Experimental procedures
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precipitation by adding cold methanol (4 × the sample volume) Purification of sulfatase
for 15 min at 0 °C. After centrifugation (13780 g for 5 min), the
The use of a promiscuous sulfatase is critical to the
development of this new mass spectrometric method.
Sulfatases usually have narrow substrate specificities and are
only commercially available in low quantities.15 This is in stark
contrast to proteomics and nucleic acid research where
proteases and phosphatases have been used to cleave even
complex natural modifications and have found general
application in their respective omics-field.16-18 We identified a
commercially available arylsulfatase (ArS) from the snail Helix
pomatia that fulfils our requirements.19 This enzyme is
commonly used in drug metabolism studies and doping
analysis investigations due to its lack of substrate selectivity.20
However, no sequence information or molecular weight has
yet been reported, which prohibits standard overexpression of
the recombinant enzyme. Direct application of this sulfatase is
impossible as it is only commercially available as a crude
extract with glucuronidase and oxidase activity. Despite this
high heterogeneity, we assessed the sulfatase activity and
substrate promiscuity by treating five synthesized and six
commercially available substrates with the crude extract. We
selected a number of structurally different sulfate esters (Fig.
2B). Our mass spectrometric analysis (ultra-high-performance
liquid chromatography tandem-mass spectrometric / UPLC-
supernatant was collected and dried in vacuo. Afterwards, the
remaining pellet was dissolved in 150 µl of water/acetonitrile
(95/5, v/v), vigorously shaken for 30 s and then centrifuged
(13780 g for 5 min). Each supernatant was collected and
transferred to
a HPLC vial for UPLC-MS/MS analysis,
alternating injection of control and assay samples to avoid
biased results.
Results and discussion
MS/MS) revealed
a wide range of sulfated substrates
hydrolyzed by this arylsulfatase (Fig. S1). Most sulfated
phenolic compounds and other aromatic alcohols were
hydrolyzed after 24 h using 130 U sulfatase, while alkyl sulfates
were almost unmodified (Fig. S1). These results confirm the
substrate promiscuity of the enzyme and its preferential for
cleavage of arylsulfate esters.
Encouraged by the substrate promiscuity of the
arylsulfatase, we performed a non-tagged enzyme purification
of the commercially available crude mixture. Throughout the
four-step chromatographic purification, we tested the
substrate specificity of all fractions in order to select those
with high sulfatase and reduced glucuronidase activity as well
as to evaluate the stability of the sulfatase during the
purification process (See SI for details). 4-Methylumbelliferyl
Fig. 1
A) Structures of the substrates used in the sulfatase / glucuronidase
comparative assay: 4-methylumbelliferyl sulfate (4) and N-acetyltyramine-O,β-
glucuronide (5), and in the units calculation assay: phenolphthalein (6) and
phenolphthalein β-D-glucuronide (7); B) SDS-PAGE of the crude H. pomatia lysate and
the purified arylsulfatase (ArS); C) Glucuronidase activity calculated for the H. pomatia
crude mixture (Sigma-Aldrich) and the purified arylsulfatase fraction.
sulfate (4) and N-acetyltyramine-O,β-glucuronide (5) were
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J. Name., 2013, 00, 1-3 | 3
Fig. 2
A) Hydrolysis experiments of selected sulfated substratePs ulesinagspeurdifioednsuolftataasdejiun sdtupmlicaaterg(Eirnrosr bars: SEM); B) Sulfated metabolites tested as substrates in the
sulfatase assay. Microbiota-derived metabolites are highlighted in red.

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selected to test sulfatase and glucuronidase activity, metabolic products of interspecies co-metabolism and the
respectively (Fig. 1A). Analytical gel filtration of the purified highest concentration of phase II metabolites of any human
arylsulfatase yielded a single peak consistent with a monomer specimen. This sample type has previously been analyzed to
in solution and a molecular weight of approximately 75 kDa as track human host and microbiota tryptophan-related
determined by SDS-PAGE (Fig. 1B and S2). The purification metabolism.21 We designed our sulfatase-based assay as
procedure yielded the arylsulfatase in the required purity for depicted in Scheme 1. Proteins were precipitated from the
our targeted metabolic analysis of human samples. urine samples using methanol to eliminate any background
Glucuronidase units activity was calculated following the enzymatic activity. Each urine sample was divided into two
described protocol in the SI using phenolphthalein (
6) and equal parts. One part was treated with purified arylsulfatase
phenolphthalein β-D-glucuronide (7). Glucuronidase and the second part with heat-inactivated arylsulfatase, which
background activity was reduced to less than 0.08% compared was used as background reference to exclude any signals not
to the activity of the crude enzyme mixture (Fig. 1C). resulting from enzymatic treatment, simplifying data analysis.
Importantly, this depleted activity does not interfere with our We treated human urine samples with sulfatase (11 U) for 17 h
Scheme 2
Workflow for our described sulfatase-based mass spectrometric strategy for the analysis of human samples. Structure validation of ferulic acid 4-sulfate (3) is
presented as an example. (Sulfatase crystal structure15
)
analysis.
to maximize hydrolysis of sulfated metabolites. We analyzed
Upon purification of the arylsulfatase, we treated a mixture each of the treated and untreated sample six times by UPLC-
of ten sulfate esters for 24 h using 19 U of sulfatase, including MS/MS in positive and negative analysis modes (Scheme 2).22
sulfated phenolic compounds [p-cresyl sulfate (
4-sulfate ), 4-methylumbelliferyl sulfate
acetyltyramine-O-sulfate ( ), -tyrosine-O-sulfate (
3-sulfate (10)], sulfated indole derivatives [indoxyl sulfate (
N-acetylserotonine-O-sulfate 11)], and alkyl sulfates
mannose 6-sulfate
13)](Fig. 2). We obtained complete sulfate hydrolysis for six features in the control group in which the sulfate esters are
metabolites after 4 h incubation time (Fig. 2A and S3). While unmodified. Over 3620 altered mass spectrometric features
two arylsulfate substrates [ -tyrosine-O-sulfate ( ) and N- (fold-change >1.5, p-value <0.0001 and intensity >30000 ion
acetylserotonine-O-sulfate 11)] were not hydrolyzed count) were identified in negative mode analysis. To
2
), ferulic acid These sample sets were compared using the XCMS
), N- metabolomics software package under R to perform peak
), estrone- detection and chromatographic alignment.23, 24
(
3
(4
8
L
9
1
),
-
We obtained clear separation of both sample sets in a
principle component analysis (Fig. S4). Our detailed data
(
[
D
(
12), taurolithocholic acid 3-sulfate analysis was focused on the identification of upregulated
(
L
9
(
completely after 4 h, they did exhibit conversion levels of 35% specifically analyze sulfates, we searched for features with a
and 45% after 24 h, respectively, which is sufficient for difference in m/z of 79.9568 Da (the difference between a
detection of the sulfate ester and identification of its structure sulfated metabolite and the corresponding hydrolyzed
in our targeted metabolomics approach.
product) within a 10 ppm limit and obtained a total of 261
features as potential sulfated metabolites. We then applied
selective MS/MS fragmentation to all 261 metabolites, which
Identification of metabolites in urine samples
After optimization of the hydrolysis conditions, we applied confirmed the presence of a sulfate ester in 188 metabolites.
this methodology to urine samples, which contain the final The total number of sulfated metabolites increased to 206
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with metabolites identified in positive mass spectrometric
Our targeted analysis of these samples led to the
mode analysis. This is about three times higher than the 65 unambiguous identification of 41 sulfated metabolites (Table
sulfated metabolites reported in the Human Metabolome S4), including the microbiota-derived metabolites p-cresyl
Database (HMDB), which demonstrates the strength of our sulfate (
2), ferulic acid 4-sulfate (3) and L-tyrosine-O-sulfate
selective method towards the identification of unknown ). These identified sulfated metabolites can either be
(
9
metabolites.25 We derived the chemical formula and compared produced by i) human metabolism only, ii) food derived
the MS/MS fragmentation for each metabolite with either metabolites, iii) co-
synthetic standards or databases (HMDB and Sirius) to
Table 2 Identified sulfated metabolites derived from microbial metabolism and
link to disease development.31-36
determine the molecular structure. Among the top hits, we
readily identified the two most
Microbial metabolite
Link to disease
Chronic kidney disease (CKD)
Cardiovascular diseases
CKD
Table 1 Number of identified sulfated metabolites throughout the selection
process (Detailed lists are presented in Tables S1-S3).
Indoxyl sulfate (
1
)
p-Cresyl sulfate (
2
)
Urine sample analysis
Feature No.
Identified features
3620
Ferulic acid 4-sulfate (
3
)
Thrombosis
and
Potential sulfated metabolites
MS2-validation of sulfate ester
Identified by MS2 fragmentation
Identified by synthetic internal standard
261
206
36
artherosclerosis prevention
Alzheimer disease
Parkinson disease
Caffeic acid 4-sulfate (14
)
Vanillic acid 4-sulfate (15
)
8
3-Hydroxyphenyl-propionic acid Parkinson disease
sulfate (16
4-Ethylphenyl sulfate (17
)
)
Autism
commonly reported microbiota-derived metabolites, indoxyl
sulfate ( ) and p-cresyl sulfate ( ). Out of the identified 206
1
2
sulfated metabolites, we validated the chemical structure of
36 metabolites including eight, which were chemically
synthesized for the unambiguous determination of their
structure (Table 1 and Tables S1-3).
As an example, we highlight the structure validation
procedure for p-cresyl sulfate (2) using a synthetic standard
(Fig. 3). Each of these molecules was synthesized and co-
injected for UPLC-MS analysis to confirm the correct chemical
structure. The extracted ion chromatograms (EIC) of the
natural and synthetic metabolite overlapped perfectly and
eluted with the same retention time. Furthermore, spiking of
the synthetic metabolite into the urine sample revealed a
single peak (Fig. 3A). Mass spectrometric fragmentation
experiments demonstrated the same fragments and
fragmentation pattern for the synthesized and natural
metabolite (Fig. 3B).
Fig. 3
Example of sulfate ester validation in urine with p-cresyl sulfate (2). A)
Identification of metabolites in fecal samples
UPLC-MS chromatogram comparison of urine sample vs. the synthesized standard
molecule; B) MS/MS fragmentation pattern of 2 in urine sample vs. the synthesized
standard molecule at a collision energy at 30 V; C) Assignment of MS/MS fragments of
2.
To investigate sulfate-dependent gut microbiota
metabolism prior to absorption by the human host, we
analyzed five different fecal samples. Analysis of this sample
type is crucial as the gut is inhabited by the largest microbial
community in the human body.26, 27 The virulence of certain
pathogenic bacteria has been linked to metabolite sulfation
and most bacterial genomes contain genes for both sulfatases
metabolism, or iv) merely by microbiota metabolism. Our
findings distinctly demonstrate the sensitivity of our targeted
method as we identified a high number of sulfated metabolites
in fecal samples, a sample set not commonly analyzed for the
detection of phase II metabolites.
29
and sulfotransferases.28, We utilized a similar workup and
analysis procedure as detailed for urine samples with minor
changes for the feature selection criteria and sample
preparation (See SI for details). To examine if the enzymatic
activity is affected by this sample type, we spiked the four
representative metabolites 1-4 into fecal sample extracts. All
four compounds were completely converted in this test assay
and proved full activity of the enzyme.
In this interdisciplinary study, we confirmed 36 metabolites
using
a combination of chemical synthesis and mass
spectrometric fragmentation experiments. These molecules
included at least seven microbiota-derived metabolites, which
have been reported to directly impact human physiology
(Table 2).30 Indoxyl sulfate (
1) and p-cresyl sulfate (2) are well
known biomarkers of chronic kidney disease and
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32
cardiovascular diseases, respectively.31,
Microbiota- D.G. gratefully acknowledges financial support from the
produced ferulic acid has been reported to prevent thrombosis Swedish Research Council (VR 2016-04423), Carl Tryggers
and artherosclerosis.33 We also identified the sulfates of Foundation (CTS 2016:155) and the Science for Life
caffeic acid (14), vanillic acid (15) and 3-hydroxyphenyl- Laboratories. S.D. received financial support from the Swedish
propionic acid (16), which have been associated with the Research Council (VR 2015-04568), the Knut and Alice
neurodegenerative Parkinson’s and Alzheimer’s diseases.34, 35 Wallenberg Foundation (WAF 2014.0183), and the European
Another connection between microbiota-derived sulfated Research Council (ERC Starting Grant 714068). We thank Erik
metabolites and human disease is the link between 4- Bivehed and Malin Nilsson Broberg for initial work and the
ethylphenyl sulfate (17) and autism.36 To our best knowledge, staff at Karolinska Institute hospital for sample collection.
comprehensive detection and analysis of these disease-related
metabolites in a single study has not yet been reported. This
Conflicts of interest
profound metabolic correlation with human health
demonstrates the importance of discovering unknown
metabolites to evaluate their impact on disease development.
Interestingly, we found several unreported sulfated
metabolites, which have only been previously detected as
their unsulfated counterparts (Tables S1 and S2). In total, 9 out
of these metabolites have previously been reported as their
unsulfated corresponding metabolites including the two
examples chavicol sulfate and 2-methoxy-4-vinylphenol
sulfate. These metabolites require further investigation to
identify their role in microbial virulence and impact on human
health.
There are no conflicts to declare.
Notes and references
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The discovery of more than three times higher number of
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Conclusions
In summary, we have established a new method for
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investigation also led to the discovery of a large number of
previously unidentified and unreported metabolites, which
represents a tremendous potential for the identification of
unknown mediators of disease development. Further
investigation of these metabolites will allow the crucial
communication between microbiota and the human host to be
deciphered. Our targeted enzymatic method is a new asset,
which expands the scope of metabolite analysis in human
samples. This strategy can be applied to metabolomics-driven
biomarker discovery for diseases affected by an altered
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