KONAN UNIVERSITY
Determination of D-Serine in Several Model
Organisms Used for Metabolic, Developmental
and/ or Genetic Researches by Liquid
Chromatography/ Fluorescence Detection and
Tandem Mass Spectrometry
著者（英）
journal or
publication title
volume
number
page range
year
URL
Masashi Syuto, Takehiro Kusakabe, Daisuke
Honda, Yo-hei Watanabe, Kojiro Takeda, Osamu
Tanaka, Hiroyuki Imai
Memoirs of Konan University. Science and
engineering series
60
1
11-19
2013
http://doi.org/10.14990/00000187
Mem. Konan Univ., Sci. & Eng. Ser., 60
（1）
11～19（2013）
11
Determination of D-Serine in Several Model
Organisms Used for Metabolic, Developmental and/
or Genetic Researches by Liquid Chromatography/
Fluorescence Detection and Tandem Mass
Spectrometry
Masashi Syuto1, Takehiro Kusakabe1,2, Daisuke Honda1,2, Yo-hei
Watanabe1,2, Kojiro Takeda1,2, Osamu Tanaka1 and Hiroyuki Imai1,2†
1
Department of Biology, Graduate School of Natural Science, Konan University, Kobe
658-8501, Japan
2
Institute for Integrative Neurobiology, Konan University, Kobe 658-8501, Japan
†
To whom correspondence should be addressed.
Fax: +81-784352539; E-mail; [email protected] (H. Imai)
(Received December 24, 2013)
Abstract
D -Amino
acids are the enantiomers of L -amino acids, and the amounts of their
D-enantiomers
are usually very low in living organisms. In the present study, the
levels of D-serine (Ser) were determined from several model organisms, and a two-step
high performance liquid chromatography method was developed using an octadecyl
silane column and a Pirkle-type chiral column after pre-column derivatization with
4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). We confirmed the presence of D-Ser
in the samples from Arabidopsis thaliana, Ciona intestinalis, Aurantiochytrium
limacinum and Parietichytrium sarkarianum, whereas D -Ser was not detected
in proteobacteria, Escherichia coli and Agrobacterium tumefaciens (Rhizobium
radiobacter). For the liquid chromatography mass spectrometry analysis, a mobile
phase containing 5 mM ammonium acetate in the mixed solution of methanol/
acetonitrile/ formic acid was selected for the resolution of NBD-Ser enantiomers
with the Pirkle-type chiral column. The current method achieved high-throughput
profiling of Ser enantiomers, which succeeded to quantify very low amounts of D-Ser
in biological samples.
Key words: Arabidopsis thaliana, Aurantiochytrium limacinum, Ciona intestinalis,
D-serine,
NBD-F, Parietichytrium sarkarianum, Pirkle-type chiral stationary phase
12
Masashi Syuto, Takehiro Kusakabe, Daisuke Honda, Yo-hei Watanabe,
Kojiro Takeda, Osamu Tanaka and Hiroyuki Imai
Introduction
D-Amino
acids are the enantiomers of L-amino acids. L-amino acids are normally
present in living organisms as the components of protein, whereas the amounts of
their D-enantiomers are usually at trace levels in animals and plants. D-Serine (Ser),
however, has been found in high levels in the brain (Hashimoto et al. 1992) and is
shown to mediate a variety of physiological N-methyl D-aspartate receptor-dependent
events (Wolosker et al. 2008). To determine D-amino acid contents, reversed-phase
high performance liquid chromatography (HPLC) with chiral derivatization reagents
such as o-phthaldialdehyde (OPA) together with N-acetyl-L-cysteine (Aswad 1984,
Gogami et al. 2006), N-isobutyryl-L-cysteine (IBLC) or N-isobutyryl-D-cysteine (IBDC)
(Brückner et al. 1994) has been reported. In addition, N-tert-butyloxycarbonyl-Lcysteine, (1)-1-(9-fluorenyl) ethyl chloroformate and the Marfey’s reagent (1-fluoro2,4-dinitrophenyl-5-L-alanine amide) were used as derivatization reagents as well.
A gas chromatography method using chiral stationary phase was also developed
(Brückner and Schieber 2000, Brückner and Westhauser 2003). In a plant study by
liquid chromatography mass spectrometry (LC-MS) with a variant of the Marfey’s
reagent, it has been reported that D-Ser inhibits the growth of Arabidopsis seedlings
at submillimolar concentration (Gördes et al. 2001). However, the determination of
small amounts of D-amino acids is still difficult tasks for complex biological samples.
Recently, a two-dimensional (2D) HPLC method combining a microbore-octadecyl
silane (ODS) column and a Pirkle-type chiral column after pre-column derivatization
with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for these issues
(Miyoshi et al. 2011). According to the reversed-phase and enantiomer resolutions
of NBD-Ser, we present here determination of D -Ser contents in a model plant
Arabidopsis thaliana as well as several model organisms by pre-column derivatization
of NBD-F.
NBD-F is a fluorescent derivatization reagent that was originally developed
for amino acid analysis (Fig. 1) (Imai and Watanabe 1981, Miyoshi et al. 2012), and
recently applied to the analyses
of other compounds such as
sphingolipid long-chain bases
(Ishikawa et al. 2013), N-methylD -aspartic
acid (Koga et al. 2012)
and glutathione (Wang et al.
2012). The use of NBD-F appears
to have several advantages that
derivatization procedure is simple
and its derivatives are highly stable
Fig. 1. Structures of D- and L-Ser and NBD-F.
(Miyoshi et al. 2012). In addition,
D-Serine in Model Organisms
13
we developed ESI-MS/MS detection of NBD-Ser derivatives, which achieved highly
selective and sensitive detection.
Materials and Methods
Chemicals and Organisms
Except where noted, all chemicals were of LC-MS grade or highest grade available
from Wako Pure Chemical (Osaka, Japan). NBD-F was purchased from Dojindo
Laboratories (Kumamoto, Japan). Seeds of Arabidopsis thaliana (Col) were
germinated in a Jiffy-7 peat pellet (42 mm i.d., Jiffy Preforma Production, Yokohama,
Japan) and grown for eight weeks under a light/dark cycle of 16/8 h (60 µmol/m2/
s) at 22 oC and then harvested for leaves and flowers. Mature adults of the ascidian
Ciona intestinalis were collected from a harbor in Kobe, Japan, or obtained from
the National BioResource Project (NBRP) and maintained as described previously
(Kusakabe et al., 2012). Whole body and neural complex (central nervous system) were
dissected and collected on ice, briefly washed with artificial seawater, and then frozen
with liquid nitrogen. The frozen tissues were stored at -80°C until use or immediately
used for the analysis. Each of Aurantiochytrium limacinum SR21 (Honda et al. 1998,
Yokoyama and Honda 2007a) and Parietichytrium sarkarianum SEK 364 (Yokoyama
et al. 2007b) was inoculated into a 500-ml conical flask containing 250-ml culture
medium (1% glucose, 0.5% poly-peptone and 0.25% yeast extract in a half of the salt
concentration of artificial seawater, Marine Art SF-1, Osaka Yakken, Japan) and
then incubated with orbital shaking for 6 days. Each of Escherichia coli JM109 and
Agrobacterium tumefaciens (Rhizobium radiobacter) GV3101was incubated into a 5-ml
tube containing LB medium for 17 h.
Preparation of Free Amino Acids and Formation of NBD Derivatives
Free amino acids were extracted by addition of 10 volume of ice-cooled methanol
(MeOH): MilliQ water (1:4, v/v). After centrifugation for 15 min at 15,000g, the
supernatant was ultrafiltrated through a 3 kD cutoff filter (Microcon Ultracel YM3, Millipore, Billerica, MA, USA). The filtrate was subjected to derivatization
with NBD-F. Aliquots of the 15,000g supernatant were used for Bradford assay to
determine protein concentration with bovine serum albumin (BSA) as a reference
protein. A 20-μl aliquot of the filtrate was mixed with 12.5 μl of 1 mg/ml NBD-F in
acetonitrile (MeCN) and 20 μl of 0.1 M borate buffer (pH 8.0) and incubated for 5
min at 65 oC. After cooling on ice for 10 min, 50 μl of 50 mM HCl was added, and the
reaction mixture was subjected to HPLC analyses.
HPLC analyses
The Ser enantiomers were analyzed by two steps of HPLC. First chromatographic
14
Masashi Syuto, Takehiro Kusakabe, Daisuke Honda, Yo-hei Watanabe,
Kojiro Takeda, Osamu Tanaka and Hiroyuki Imai
resolution was performed using a TSKgel ODS–100Z column (250 mm x 2.0 mm i.d.,
Tosoh Bioscience, Tokyo, Japan) held at 30 oC. For each run, 1–5 μL of each sample
was injected. A binary elution gradient consisting of 0.05% (v/v) trifluoroacetic acid
(TFA) in water as solvent A and 0.05% (v/v) TFA in MeCN as solvent B was used.
The flow rate was 200 μL/min with the following conditions: 75% solvent A /25%
solvent B initially, maintained for 15 min, then proceed to 100% solvent B in a linear
fashion after 15 min, and maintained with 100% solvent B for an additional 5 min.
The target amino acid fractions were collected and dried under reduced pressure at
25 oC. The residue was dissolved with MeCN and subjected to 2nd HPLC analysis.
Chromatographic resolution was performed using a SUMICHIRAL OA-2500S column
(2 mm i.d. x 250 mm, Sumika Chemical Analysis Service, Tokyo, Japan) held at 30 oC
(Miyoshi et al. 2011). The mobile phase was 50 mM ammonium acetate in the mixed
solution of MeOH/ MeCN (1/1, v/v). For each run, 1–5 μL of each sample was injected,
and the flow rate was 200 μL/min. Flow from the columns was detected with a RF10AXL fluorescence detector (Shimadzu, Kyoto, Japan). The fluorescence of the NBD
derivatives was measured with an excitation wavelength of 470 nm and an emission
wavelength of 530 nm. NBD derivatives of D- and L-Ser were identified by comparing
their retention times and MS/MS spectra of product ion scanning with those of
commercially-obtained authentic standards.
For the LC–MS/MS analysis, flow from the SUMICHIRAL OA-2500S column
was detected with an ACQUITY TQD tandem quadrupole mass spectrometer (Waters,
Milford, MA, USA). The mobile phase was 5 mM ammonium acetate in the mixed
solution of MeOH/ MeCN/ formic acid (2000/2000/1, by volume). The transitions of the
precursor ions [M + H]+ to a main product ion m/z 223 were used as precursor/product
ion pairs in the positive ionization multiple reaction monitoring (MRM) mode (Song et
al. 2007). The following conditions were used: capillary voltage, 3 kV; desolvation gas
flow, 600 L/h; nebulizer gas flow, 50 L/h; source temp., 120 oC; and collision gas flow, 0.3
mL/min (4–5 mbar).
Results and Discussion
For selective determination of D-Ser in various samples with minimal interference,
two-step HPLC procedures using an ODS column and an enantioselective column
have been developed. Amino acid samples filtrated through a 3 kD cutoff filter were
pre-column derivatized with NBD-F and separated by a high carbon content ODS
column (2.0 mm i.d. x 250 mm), and an aqueous solution containing 25% MeCN and
0.05% TFA was selected as the mobile phase for the appropriate retention of NBDSer. In the present study, NBD-derivatives of D- and L-Sers were prepared from leaves
and flowers (10 mg fresh weight of each) of Arabidopsis thaliana. As shown in Fig. 2a,
a good resolution of chromatographic peaks was observed using a TSKgel ODS–100Z
D-Serine in Model Organisms
15
Fig. 2. ‌Reversevd-phase and enantiomer resolutions of NBD-Ser. (a) A chromatogram of
NBD-Ser in Arabidopsis flowers obtained by reversed-phase resolution with a TSKgel ODS column. (b) A chromatogram of NBD-Ser in Arabidopsis flowers obtained by
enantiomer resolution using a Sumichiral OA-2500S column. The fractions indicated
by a thick bar were collected and subjected to the Sumichiral OA-2500S column. The
chromatographic peaks were identified by comparing the retention time and MS/MS
spectra of product ion scanning with those of the authentic standard.
column (2.0 mm i.d. x 250 mm); NBD-Sers was detected at around 8 min. For the
second column chromatography, NBD-D-Ser and NBD-L-Ser were separated using a
Pirkle type enantioselective column (Sumichiral OA-2500S, 2.0 mm i.d. x 250 mm).
The mobile phase was selected to obtain the appropriate retention of NBD-Sers: 50
mM ammonium acetate in the mixed solution of MeOH/ MeCN (1/1, v/v) was used.
As shown in Fig. 2b, the present HPLC condition enabled the sufficient resolution of
NBD-D-Ser and NBD-L-Ser.
Table 1. Content levels of D- and L-serine enantiomers in model organisms
A. thaliana
flower
leaf
C. intestinalis
neural complex
whole body
A. limacinum
P. sarkarianum
E. coli
A. tumefaciens
SR21
SEK364
JM109
GV3101
(nmol/ mg soluble protein)
D-Ser
7.64
0.15
1.80
0.026
9.20
9.31
n.d.
n.d.
L-Ser
29.3
90
10.9
0.083
11.0
38.6
4.70
3.19
D/D+L
20.7
0.17
14.2
23.9
45.5
19.4
n.d.
n.d.
(%)
n.d., not determined: the limit of detections (LODs) of the present methods were approximately 10 attomole/ injection.
Indispensable amounts of D-Ser were found in the samples from A. thaliana,
C. intestinalis, A. limacinum and P. sarkarianum (Table 1). In A. thaliana, relatively
large amount of D-Ser was found in flowers (7.64 nmol/ mg soluble protein), which
may stimulate further investigations on the physiological functions of D-Ser in plant
sexual reproduction. Recently, it has been shown in A. thaliana that D-Ser activates
glutamate receptor-like channels in the apical region of pollen tubes, allowing Ca2+
permeation into the cytoplasm (Michard et al. 2011). In C. intestinalis, the level of
Masashi Syuto, Takehiro Kusakabe, Daisuke Honda, Yo-hei Watanabe,
Kojiro Takeda, Osamu Tanaka and Hiroyuki Imai
16
D-Sers
was 692-fold higher in neural complex than that in whole body. High levels
of D-Ser to the total Ser were found in marine labyrinthulids, A. limacinum and P.
sarkarianum, with large amounts (45.5 and 19.4% of the total Ser in A. limacinum and
P. sarkarianum, respectively). Interestingly, D-Ser was not detected in proteobacteria,
E. coli and A. tumefaciens (Rhizobium radiobacter).
It has been reported that teicoplanin aglycone chiral stationary phase offers
a good resolution for NBD-Ser enantiomers with a mobile phase consisting of MeOH/
water/TFA (60/ 40/ 0.012, by volume) (Song et al.2007). In contrast, resolution of
NBD-Ser enantiomers on Pirkle-type chiral stationary phases has not previously
been reported in the LC-MS/MS analysis probably because mobile phases used for the
Pirkle-type chiral stationary phases were not compatible with MS detection or not
good enough to resolve the amino acid enantiomers. In the present study, we prepared
a mobile phase that contains 5 mM ammonium acetate in the mixed solution of MeOH/
MeCN/ formic acid (2000/2000/1, by volume). In product ion scanning of NBD-D-Ser,
the transition of precursor ions [M+H]+ to the product ion was detected at m/z 223 at
a comparable intensity (Fig. 3a). In addition, the MRM chromatogram of the transition
of [M+H]+ to m/z 223 exhibited the sufficient resolution of the NBD-D-Ser and NBDL-Ser
(Fig. 3b). These improved peak resolution contributed to the high selectivity
and sensitivity of our present method capable of quantifying several fmol of analytes
injected on column, although the retention times of the NBD-Ser enantiomers were
very long at 1 h; the retention times will be minimized in the future developments on
this system.
Fig. 3. ‌Analysis of NBD-Ser by LC-MS/MS. (a) Mass spectra of NBD-D-Ser. A standard of
D-Ser was applied to ESI-MS/MS. The panel (a) shows fragmentation patterns of
NBD-D-Ser which were performed as product ion scanning targeted for [M+H]+. (b)
An ion chromatogram for the transition of [M+H]+ to 223 was obtained by enantiomer
resolution using a Sumichiral OA-2500S column. A mixture of D- and L-Sers was
applied to the column.
D-Serine in Model Organisms
17
In conclusion, we here developed HPLC-fluorescence detection/ ESI-MS/
MS of Ser enantiomers using NBD derivatives, which succeeded to quantify very low
amounts of D-Ser in biological samples. The current method should be applicable
to seeking the active components of D -amino acids from various organisms for
assessment of physiological status in living organisms.
Acknowledgments
We thank Megumi Mukai for experimental assistance. Parts of this work were
supported by Special Ordinary Expense Subsidies for Private Universities from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
This research was also supported in part by the Strategic Research Foundation Grantaided Project for Private Universities from MEXT of Japan. H.I. thanks Masashi Mita,
Frontier Science Business Division, Shiseido Co., Ltd., for helpful discussions.
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