特集 SFC バイオの軌跡［招待論文：総説・レビュー論文］

A Reflection on Two Decades ofMetabolomic Innovations in Capillary
Electrophoresis-Mass Spectrometry at
the Institute for Advanced Biosciences,
Keio University
メタボロミクスの技術革新に対する慶應義塾大学先端生命科学研究所の 20 年に亘る貢献
Philip Britz-McKibbin

Professor, Department of Chemistry and Chemical Biology, McMaster University, Hamilton,
Canadaフィリップ ブリッツ - マッキビンマックマスター大学（カナダ）化学・ケミカルバイオロジー学科教授
Correspondence to: britz@mcmaster.ca

Abstract:

The Institute for Advanced Biosciences (IAB) at Keio University was firstestablished in April 2001 to become a leading international metabolomics center foradvancing integrated systems biology research. Unlike other metabolomic centersworldwide, IAB strategically focused on developing capillary electrophoresismass spectrometry (CE-MS) technology as their central instrumental platform forcomprehensive analysis of polar/ionic metabolites in conjunction with bioinformatic
tools for biomarker discovery and biochemical interpretation. In addition to hosting
two International Metabolomics Society conferences, training young scientists
and founding successful spin-off companies, IAB has contributed to high impact
metabolomic studies as applied to clinical medicine, functional genomics and multiomic studies, cancer diagnostic testing, agriculture and food science, as well as
epidemiology and population health. IAB has also fostered metabolomic partnerships
with the local community, clinicians and industry as reflected by their involvement
in a prospective study of community-dwelling residents from Tsuruoka City andassessment of the quality of foods produced from the Shonai area. IAB has also
spearheaded numerous CE-MS based metabolomic research collaborations elsewhereacross Japan and worldwide. This short review will strive to highlight key milestones
spanning over 20 years of research innovations in CE-MS metabolomics technology
brought forth under the leadership of Dr. Masaru Tomita.

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【第 1 章 先端生命科学】
A Reflection on Two Decades of Metabolomic Innovations in Capillary ElectrophoresisMass Spectrometry at the Institute for Advanced Biosciences, Keio University
慶應義塾大学先端生命科学研究所 (IAB) は 2001 年 4 月に設立され、統合システム生物学研究を推進するための国際的なメタボロミクスセンターとしての役割を果たしている。世界中の他のメタボロミクスセンターとは異なり、IAB は極性・イオン性代謝物の包括的な解析プラットフォームとしてのキャピ
ラリー電気泳動質量分析（CE-MS）技術の開発と、バイオマーカー探索や生
化学的解釈のためのバイオインフォマティックツールの開発に重点を置いて戦略的に行ってきた。IAB は 2 回の国際メタボロミクス会議の主催、若手科
学者のトレーニングやスピンオフ企業の設立に加えて、臨床医学、機能ゲノミ
クス、マルチオミクス研究、がん診断検査、農業および食品化学、疫学や公衆衛生学等の、インパクトの大きなメタボロミクス研究に貢献してきた。IAB はま
た、鶴岡市の地域住民を対象とした前向き研究の実施や庄内地域で生産された食品の品質評価に代表されるように、地域社会、臨床医や産業界とのメタボ
ローム研究を介したパートナーシップを育んできた。さらに、IAB は日本および世界の数々の CE-MS を用いたメタボローム共同研究の先頭に立ってきた。
この短いレビューでは、冨田勝博士のリーダーシップの下でもたらされた 20
年以上に亘る CE-MS メタボロミクス技術の研究イノベーションの主要な成果について紹介する。

Keywords: metabolomics, capillary electrophoresis-mass spectrometry (CE-MS), non-targeted
metabolite profiling, biomarker discovery, clinical medicine

メタボロミクス、キャピラリー電気泳動－質量分析（CE-MS）、非標的代謝物プロファイリング、バイオマーカー探索、臨床医学

1 stablishing CE-MS as a Robust Platform for Metabolomics:
E
The Early Years
The principles of biochemical individuality and disease susceptibility (Garrod,

1902; Williams, 1956) may be revealed via quantitative metabolite profiling (Horning& Horning, 1971; Pauling et al., 1971), which can be traced back over the past
century. However, a renaissance in metabolism was sparked more than twenty years
ago when metabolomics was proposed as a functional genomics tool to understandthe role of gene mutations on molecular phenotype with the advent of large-scale
genomic sequencing projects (Oliver et al., 1998; Fiehn et al., 2000). Contemporary
metabolomics research has relied on recent advances in analytical instrumentationand computational analysis, including high-resolution mass spectrometry (MS) andhigh-field nuclear magnetic resonance (NMR) infrastructure (Kuehnbaum & BritzMcKibbin, 2013). For instance, gas chromatography-mass spectrometry (GC-MS)
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and increasingly liquid chromatography-mass spectrometry (LC-MS) using reversedphase and/or hydrophilic interaction modes of separation are widely used inmetabolomic studies. Alternatively, capillary electrophoresis-mass spectrometry (CEMS) oﬀers a high eﬃciency microseparation platform ideal for characterization of
the ionic metabolome from volume- or mass-limited biospecimens, such as residual
infant sweat (Nori de Macedo et al., 2017), a single dried blood spot punch(DiBattista et al., 2019), or a lyophilized muscle tissue biopsy (Saoi et al., 2019). Dr.
Masaru Tomita early on recognized the potential of CE-MS technology formetabolomics since the majority of primary metabolites within cells are hydrophilic/charged ions, whose measurements could be used to simulate cell behavior in silico
for deeper insights into dynamic metabolic processes critical to life (Tomita et al.,1999).
In 2003, researchers from IAB at Keio University first reported a methodology
for untargeted metabolite profiling using CE-MS allowing for determination of 352
metabolite standards, along with characterization of a total of 1692 known andunknown metabolites from B. subtilis extracts (Soga et al., 2003). In this case, acoaxial sheath liquid interface was used to couple CE with a time-of-flight (TOF)
mass spectrometer, which was operated initially under three distinct modes as
depicted in Figure 1. This included an acidic buﬀer (1.0 M formic acid, pH 1.8) for
separation of cationic metabolites in an unmodified fused-silica capillary underpositive ion mode, an alkaline buffer (50 mM ammonium acetate, pH 8.5) for
separation anionic metabolites using a cationic polymer coated capillary undernegative ion mode, and a second alkaline buﬀer (50 mM ammonium acetate, pH 7.5)
for resolving highly charged nucleotides and coenzyme A metabolites with a neutral
coated capillary under negative ion mode. The latter pressure-assisted CE-MS mode
was deemed necessary due to deleterious adsorption of multivalent anions to the
cationic polymer coated capillary (Soga et al., 2002a) as electrophoretic separations
were performed under reversed polarity, such that anions co-migrate with an anodic
electroosmotic flow (EOF) to avoid deleterious current drops (Soga et al., 2002b).

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【第 1 章 先端生命科学】
A Reflection on Two Decades of Metabolomic Innovations in Capillary ElectrophoresisMass Spectrometry at the Institute for Advanced Biosciences, Keio University
Figure 1. Original CE-MS protocol used
for comprehensive metabolite profiling of
B. subtilis cell extracts performed under
three different configurations for the
analysis of (A) cationic metabolites, (B)anionic metabolites, and (C) multivalentanions, such as nucleotides and coenzyme
A metabolites (Reproduced from Soga etal., 2003).

Pressure assisted-CE-MS for analyzing multivalent nucleotides was later proposedusing a silanol masking technique by preconditioning the cationic polymer coatedcapillary with phosphate buﬀer prior with the nebulizer gas turned oﬀ to avoid ion
suppression (Soga et al., 2007). However, this approach was complicated and time
consuming since repeat preconditioning with an involatile phosphate buffer wasneeded prior to each run to obtain adequate reproducibility.
A major breakthrough to improve the long-term stability of CE-MS for anionicmetabolite profiling under reversed polarity was discovered when using a platinum
electrospray ionization spray needle (Soga et al., 2009) that addressed the poorrobustness reported in an inter-method comparison of cellular metabolites by CE-MS
relative to LC-MS and GC-MS protocols (Büscher et al., 2009). This problem was
subsequently attributed to incidental corrosion of the stainless steel sprayer in CE-MS
at the anodic electrode due to electrolysis that eventually plugged the capillary outlet
with iron oxide precipitates resulting in shortened capillary lifetimes (Soga et al.,2009) as shown in Figure 2. This new method had several advantages since it
integrated two CE-MS previous methods originally developed for anionic metabolites
under negative ion mode (Figure 1B,C) into a single robust protocol that also
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特集 SFC バイオの軌跡
Figure 2. Improved long-term robustness of
CE-MS protocol with a coaxial sheath liquid
sprayer for anionic metabolites with acationic polymer coated capillary underreversed polarity when using a (A) standard
stainless steel and a (B) platinum needle.
The greater endurance and reproducibilityof CE-MS (C) with the platinum ascompared to stainless steel needle are
evident after 541 repeated sample injections
were achieved as it avoids needle corrosion
and plugging of the capillary outlet by ironoxides due to electrolysis (Reproduced from
Soga et al., 2009).

improved sensitivity for certain metal chelating anions (e.g., citrate). As a result, two
CE-MS protocols were standardized for cationic and anionic metabolite profiling,which were implemented into future metabolomic studies at IAB. This example
highlights the importance of rigorous method optimization and validation studies
when developing reliable CE-MS methods since other operating conditions may
impact anionic metabolite profiling under negative ion mode (van Mever et al., 2019).
For instance, aminolysis of the polyimide coating of the fused-silica capillary canoccur when using strongly alkaline ammonium based volatile buffers (pH > 9.0),
which leads to incidental capillary fractures with shorter lifetimes (Yamamoto et al.,2016).
A major bottleneck when performing nontargeted metabolomics involves preprocessing of raw data files in CE-MS, which requires multiple steps for spectral
deconvolution, data filtering, data smoothing, and peak picking (Shanmuganathan etal., 2021b). Indeed, there exists a large number of poorly characterized peaks inuntargeted metabolomics using ESI-MS, including unreliable and redundant signals
generated from the same compound (Saito et al., 2021). Although there are several28

【第 1 章 先端生命科学】
A Reflection on Two Decades of Metabolomic Innovations in Capillary ElectrophoresisMass Spectrometry at the Institute for Advanced Biosciences, Keio University

open-source and vendor-independent software programs optimized for LC-MS
metabolomics (e.g., XCMS, mzMine), the distinct electromigration behavior andpeak shapes for ions in CE-MS may benefit from customized solutions than existingvendor software developed for chromatographic separations. For these reasons,researchers at IAB first developed a Mathematica software package referred to as
MathDAMP for diﬀerential analysis of metabolomic data sets to facilitate their directcomparisons for biomarker identification (e.g., disease versus control). Raw data sets
were automatically preprocessed and normalized in terms of migration times andintegrated peak areas and then visualized using density plots (Baran et al., 2006).
Later, Morohashi et al. (2007) introduced a post-measurement peak filtering method
for CE-MS, referred to as P-BOSS, as a way to prevent data overfitting due to a large
fraction of spurious signals and background peaks detected in nontargeted
metabolomic data workflows. A proprietary software referred to as MasterHands waslater developed for processing and annotating raw data generated by CE-TOF-MS(Sugimoto et al., 2010b). Furthermore, researchers at IAB have also contributed to awidely used KEGG-based pathway visualization tool for complex -omics data sets
(Arakawa et al., 2005), including a web-based metabolite pathway browser to aide in
the biochemical interpretation based on the KEGG Atlas (Kono et al., 2009). As aresult, Dr. Masaru Tomita commanded several important milestones in CE-MS for
robust methodology, software development and bioinformatic tools to enable cuttingedge metabolomics research.

2

Novel Developments in CE-MS for Improving Sensitivity andUnknown Identification
Concentration sensitivity remains a major limitation in CE-MS based

metabolomics as compared to LC-MS due to the small injection volumes introduced
on-capillary (~ 10 nL) when using a conventional coaxial sheath liquid interface as itleads to post-capillary dilution eﬀects while stabilizing spray formation (Maxwell &Chen, 2008). In this case, a sheathless CE-MS interface based on a porous sprayer
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design was evaluated for the analysis of cationic metabolites (Hirayama et al., 2012),
which was found to improve the detection limit for a subset of compounds by morethan 5-fold corresponding to a 10-fold increase metabolome coverage of urine
samples relative to a sheath flow CE-MS. However, a longstanding obstacle has been
the long-term stability and reliability of various sheathless CE-MS interface designs
that may be too fragile and expensive to routinely operate. Researchers at IAB later
developed their own customized sheathless CE-MS interface easily fabricated bymaking a small crack about 2 cm from the end of a capillary column, which was
fixed on a plastic plate with a dialysis membrane to prevent sample loss (Hirayama etal., 2018). Despite its convenience and cost-eﬀectiveness, only a modest sensitivity
gain of 2.5-fold was achieved for cationic metabolites analyzed from cancer cell
extracts as compared to a conventional sheath liquid CE-MS system. In contrast,
sheathless CE-MS together with on-line sample preconcentration via transientisotachophoresis has been reported to greatly improve concentration sensitivity by
two orders of magnitude allowing for deeper urine metabolome coverage (Ramautar
et al., 2012).
To date, nontargeted CE-MS metabolomic analyses have been largely performed
using TOF mass analyzers due to their high mass resolution (~ 40,000 full-width at
half maximum or FWHM), fast acquisition times, and favorable cost-benefit
analytical performance. Recently, a new electrospray ionization adapter wasdeveloped to couple CE with a commercial Orbitrap mass analyzer, which offers
greater mass resolution (~ 140,000 FHWM) and better sensitivity for detection oflower abundance metabolites than a conventional TOF system as depicted in Figure
3 (Sasaki et al., 2019). For the first time, CE with ultra-high resolution MS using asheath flow interface with grounded nebulizer was used to quantify over 270 cationicand anionic metabolites in plasma filtrate extracts with good linearity, accuracy andrepeatability following method validation. Importantly, a large number of bioactive
plasma peptides and their isobars were identified and resolved by this platform thathave not been previously reported. Future studies are needed to better assess the long-30

【第 1 章 先端生命科学】
A Reflection on Two Decades of Metabolomic Innovations in Capillary ElectrophoresisMass Spectrometry at the Institute for Advanced Biosciences, Keio University

Figure 3. Deeper plasma metabolome coverage by CE-MS was achieved when using acustomized sheath flow and grounded nebulizer interface as an electrospray ionization adaptor
when coupled to an ultrahigh resolution orbitrap MS system (Reproduced from Sasaki et al.,2019).

term performance of this new CE-MS platform while developing integrated software
control to improve instrument usability in metabolomic studies. Improved
concentration sensitivity can be also achieved in CE when using multiple reaction
monitoring with tandem mass spectrometry (MS/MS) that is optimal for targeted
metabolite analysis of specific metabolite classes, as well as clinical and exposure
biomarkers with lower detection limits, such as amino acids (Soga et al., 2004),
γ-glutamyl dipeptides (Saoi et al., 2020) and 1-hydroxypyrene glucuronide (Gill &Britz-McKibbin, 2020).
Apparent migration times of ions in CE are more variable than retention times
in reversed-phase LC due to changes in the EOF between runs, which can result inerroneous peak alignment and mis-annotation of metabolites from complex mixtures(Salzer et al., 2022). On the other hand, the eﬀective (or apparent) electrophoretic
mobility represents a more reliable and fundamental physicochemical property of an
ion that can also support unknown metabolite identification when using computer
simulations complementary to collision-induced dissociation tandem mass
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spectrometry (MS/MS) (Lee et al., 2007). In fact, a recent inter-laboratory ring trialof a standardized CE-MS method for analysis of 21 cationic metabolites using
diﬀerent instrumental configurations across 13 laboratories demonstrated acceptablereproducibility when measuring the eﬀective electrophoretic mobility of an ion rather
than its relative migration time normalized to a single internal standard (Drouin et al.,2020). As a result, Sugimoto et al. (2005) proposed the use of two internal standard tobetter normalize apparent migration times for cationic metabolites in CE-MS insupport of their identification among several putative isomeric and isobaric
candidates. In this case, the electromigration behavior of ions was accurately
predicted when using an artificial neural network algorithm based on their pKa (since
it reflects the eﬀective charge of an ion) as well as 152 other molecular descriptors
derived in silico from their known chemical structure. This approach correctlyclassified metabolites in 78% of cases among the top three candidate ions following a
Kyoto Encyclopedia of Gene and Genomics (KEGG) and Human Metabolome
Database (HMDB) search (Sugimoto et al., 2005). Researchers at IAB later refined
this strategy for unknown metabolite identification in CE when modeling thenormalized migration time of 375 cationic metabolites as training set while using
support vector regression to prevent model overfitting (Sugimoto et al., 2010a). Thismethod was then used to predict the normalized migration times for 2,938 polar
metabolites in CE-MS, which improved the identification rate for peaks assigned tolikely candidate ions from open-access public databases. Future work may also be
extended towards virtual quantification of recently identified metabolites when using
CE when standards are unavailable based on modeling of solute ionization responses
in ESI-MS that can vary widely for equimolar concentrations of metabolites insolution (Chalcraft et al., 2009).

3 E-MS for Large-scale Metabolomics Studies: Beyond PolarCMetabolites?
To date, there have been sparse large-scale CE-MS metabolomic studies given32

【第 1 章 先端生命科学】
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lingering concerns of long-term instrumental stability and robustness over several
years of prospective laboratory analyses (Belczacka et al., 2018; Shanmuganathan etal., 2021b). Researchers at IAB have been involved with the Tsuruoka Metabolomics
Cohort Study (TMCS) comprising the recruitment of 11,002 local Japaneseparticipants aged from 35 to 74 years, which may provide new insights in dietary
exposures and lifestyle on disease risk assessment (Sasaki et al., 2018). For instance,
Harada et al. (2016) first reported the analysis of 115 polar metabolites from fasting
plasma samples using standardized CE-MS methods, which were collected from 896
Japanese men who participated in the baseline survey of the TMCS. In this work,
nineteen metabolites were associated with daily alcohol consumption which wasvalidated in an independent replication population, whereas three plasma metabolites
were associated with alcohol induced liver injury, namely threonine, glutamine, andguanidinosuccinate (Harada et al., 2016). A follow-up study also identified plasma
biomarkers associated with physical inactivity from 1,193 participants in the TMCS
cohort when using CE-MS as it is a modifiable risk factor that increases chronic
disease burden (Fukai et al., 2016). A panel of plasma metabolites, including severalamino acids and their derivatives were associated with self-reports of sedentary
behavior when using linear regression models with covariate adjustments, including
age, body mass index, smoking, alcohol intake, and energy intake. The same groupsubsequently reported the absolute concentrations of 94 polar metabolites in 8,413
fasting plasma samples from participants in the TMCS study, which demonstrated
good intermediate precision with acceptable intra-batch and inter-batch variability(Harada et al., 2018). To accomplish this milestone, four dedicated CE-TOF-MS
instruments were used for metabolomics analyses with data acquired over 52 months(30 samples per day with QC every 10 runs) comprising 105 running batches ofcations and 99 batches of anions. An inter-laboratory method comparison was also
performed for plasma creatinine and uric acid concentrations measured by CE-MS as
compared to validated clinical enzyme assays on matching serum samples, which
demonstrated good mutual agreement with a mean bias of 10 to 13% (Harada et al.,KEIO SFC JOURNAL Vol.22 No.2 2022

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2018). A non-targeted approach was later applied to classify food intake biomarkers
in plasma from 7,012 eligible participants from the TMSC study who completed astandardized food frequency questionnaire (Shibutami et al., 2021). Overall, 21
plasma metabolites were associated with the intake of nine self-reported food groups,
such as hydroxyproline for meat, trimethylamine-N-oxide for fish, choline for eggs,
as well as quinic acid and trigonelline for coffee. A large-scale CE-MS urine
metabolomic study was also recently reported for 6,720 participants from the TMCS
cohort, which included the quantification of 123 polar metabolites (Ishibashi et al.,2021). This study highlighted that spot morning urine samples are suitable for
epidemiological studies given their consistent correlation with metabolites measured
in a sub-set of matching 24-h urine samples with acceptable reproducibility for mostcompounds as reflected by a CV < 20% measured intermittently from QC samples.
Given the lower throughput of electrophoretic and chromatographic separations
that rely on single sample injections with column reconditioning (< 30-40 min/sample), serial sample injections based on multisegment injection (MSI)-CE-MS
oﬀer a simple way to greatly boost sample throughput, encode spectral information
temporally while also ensuring better long-term data fidelity without added
infrastructure costs (Kuehnbaum et al., 2013), including a robust QC-based batchcorrection for longitudinal metabolomics data (Shanmuganathan et al., 2021a). Thisrenders MSI-CE-MS competitive with robust NMR metabolomic methods given its
greater sensitivity and metabolome coverage, smaller sample volume requirements,
as well as lower operating costs and greater sample throughput (Shanmuganathan etal., 2021a). Recently, Igarashi et al. (2021) demonstrated that up to 40 consecutive
samples can be analyzed by MSI-CE-MS in a single run with an effective sample
throughput of 1 min/sample with acceptable reproducibility and accuracy when using
matching deuterated internal standards for correction of ion suppression effects asshown in Figure 4. This approach was applied to a targeted analysis of salivary
polyamines when using CE-MS/MS that found N1-acetylspermine as a promisingbiomarker for colorectal cancer. Moreover, there was a strong correlation in salivary34

【第 1 章 先端生命科学】
A Reflection on Two Decades of Metabolomic Innovations in Capillary ElectrophoresisMass Spectrometry at the Institute for Advanced Biosciences, Keio University

Figure 4. Serial injection of 40 consecutive analyses of polyamine standards (1 mM) and theirdeuterated internal standards when using MSI-CE-MS as a high throughput platform formetabolomics. Abbreviations: DiAc, diacetyl; Spm, spermine; Ac, acetyl; and Spd, spermidine(Reproduced from Igarashi et al., 2021).

polyamine concentrations measured by MSI-CE-MS as compared to conventional
single injection CE-MS and LC-MS/MS methods (Igarashi et al., 2021). However,
reversed-phase LC-MS remains the central platform for lipidomic studies as waterinsoluble lipids are not measured by CE-MS protocols using aqueous backgroundelectrolyte systems. However, recent advances in developing multisegment injectionnonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS) offer a
rapid yet multiplexed separation platform for the nontargeted analysis of diverse
classes of ionic lipids differing in polarity in serum extracts, such as
phosphatidylcholines, phosphatidic acids, phosphatidylinositols and nonesterified
fatty acids (Azab et al., 2019; Ly et al., 2022). MSI-NACE-MS is anticipated togreatly increase overall metabolome coverage beyond polar metabolites given thelarge number of lipids present in blood specimens that requires better reporting
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standards and data harmonization (Bowden et al., 2017).

4

Conclusions and Future Perspectives
Over the past twenty year, Dr. Masaru Tomita has established an exceptional

metabolomics facility in rural and northern Japan featuring the largest number ofoperating CE-MS instruments worldwide. The development of robust andstandardized CE-MS protocols together with software packages for data preprocessing and visualization have been critical to influential breakthroughs intranslational science when using nontargeted metabolite profiling for biomarker
discovery. These include CE-MS metabolomic applications in integrative systems
biology to evaluate perturbations and multiple gene knockout mutations on E. coli
(Ishii et al., 2007; Nakahigashi et al., 2009), metabolite indicators associated withsensory evaluation scores of Japanese sake or dry cured ham (Sugimoto et al., 2010c;
Sugimoto et al., 2020), and diagnostic biomarkers for oral and colorectal cancer
screening (Ishikawa et al., 2016; Kuwabara et al., 2022). His sustained eﬀorts havealso supported local companies and community residents living in Tsuruoka citywhile fostering a diverse array of engaging collaborations from across Japan andinternationally. Moreover, several successful bio-venture spin-off companies havealso been founded based on the fundamental metabolomics research program at IAB,
including Human Metabolome Technologies Inc, Saliva Tech. Co., and Spiber Inc.
The impact of large-scale metabolomics studies to assess environmental exposuresthat modulate chronic disease risk is still underway, which may reveal preventative
dietary and lifestyle patterns to promote human health, well-being and longevity.
Future technology development in CE-MS promises to further enhance concentration
sensitivity, sample throughput, data quality, and metabolome coverage to encompass
a wide range of ionic lipids in complex biological samples.

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Acknowledgements
P.B.M. acknowledges funding from the Natural Sciences and Engineering ResearchCouncil of Canada, Cystic Fibrosis Canada, the Canada Foundation for Innovation and Genome
Canada.

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