Structural determination of cerebrosides isolated from Asterias amurensis starfish eggs using high-energy collision-induced dissociation of sodium-adducted molecules

Article abstract of DOI:10.1002/rcm.4896

Six cerebrosides were isolated from the eggs of the starfish Asterias amurensis using solvent extraction, silica gel column chromatography, and reversed-phase high-performance liquid chromatography. This study demonstrated that the structures of cerebrosides could be completely characterized, based on their sodium-adducted molecules, using fast atom bombardment (FAB) tandem mass spectrometry. The high-energy collision-induced dissociation of the sodium-adducted molecule, [M+Na]⁺, of each cerebroside molecular species generated abundant ions, providing information on the compositions of the 2-hydroxy fatty acids and long-chain sphingoid bases, as well as the sugar moiety polar head group. Each homologous ion series along the fatty acid and aliphatic chain of the sphingoid base was useful for locating the double-bond positions of both chains and the methyl branching position of the long-chain base. The N-fatty acyl portions were primarily long-chain saturated or monoenoic acids (C16 to C24) with an α-hydroxy group. The sphingoid long-chain base portions were aliphatic chains (C18 or C22) with two or three degrees of unsaturation and with or without methyl branching.

Full text of DOI:10.1002/rcm.4896

Research Article
Received: 26 September 2010
Revised: 5 November 2010
Accepted: 6 December 2010
Published online in Wiley Online Library: 2011
Rapid Commun. Mass Spectrom. 2011, 25, 572–578
(wileyonlinelibrary.com) DOI: 10.1002/rcm.4896
Structural determination of cerebrosides isolated from Asterias
amurensis starfish eggs using high-energy collision-induced
dissociation of sodium-adducted molecules
Taeseong Park1, Young Seung Park1, Jung-Rae Rho2 and Young Hwan Kim1,3,4
*
1Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang 863-883, Korea
2Deptartment of Marine Information Science, Kunsan National University, Kunsan 573-701, Korea
3Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Korea
4Department of Bio-Analytical Science, University of Science and Technology, Daejeon 305-333, Korea
Six cerebrosides were isolated from the eggs of the starfish Asterias amurensis using solvent extraction, silica gel
column chromatography, and reversed-phase high-performance liquid chromatography. This study demonstrated
that the structures of cerebrosides could be completely characterized, based on their sodium-adducted molecules,
using fast atom bombardment (FAB) tandem mass spectrometry. The high-energy collision-induced dissociation of
the sodium-adducted molecule, [M R Na]R, of each cerebroside molecular species generated abundant ions, provid-
ing information on the compositions of the 2-hydroxy fatty acids and long-chain sphingoid bases, as well as the sugar
moiety polar head group. Each homologous ion series along the fatty acid and aliphatic chain of the sphingoid base
was useful for locating the double-bond positions of both chains and the methyl branching position of the long-chain
base. The N-fatty acyl portions were primarily long-chain saturated or monoenoic acids (C16 to C24) with an
a-hydroxy group. The sphingoid long-chain base portions were aliphatic chains (C18 or C22) with two or three
degrees of unsaturation and with or without methyl branching. Copyright ß 2011 John Wiley & Sons, Ltd.
Cerebrosides are glycosphingolipids consisting of
a
useful technique for determining the complete structures of
glycerolipids, including glycoglycerolipids,[21] phospholi-
pids,[22] and triacylglycerols.[23] It was used to identify the
polar head group and fatty acid compositions, as well as
the double-bond position in the fatty acyl chains. Ann and
Adams contributed significantly to the characterization of
ceramides and neutral glycosphingolipid structures using
CID of lithium-adducted molecules.[13,19] The fragmentation
mechanisms of product ions observed in the low-energy CID
of [M þ Li]þ ions of glucosylceramides have been studied
extensively.[15] Recently, five glucosylceramides isolated
from marine sponges were characterized using FAB-linked
scanning at constant B/E.[24]
ceramide and a single sugar residue (glucose or galactose)
at C-1. The hydrophobic ceramide portion contains a
sphingoid base and an amide-linked fatty acyl chain, while
the hydrophilic portion is a sugar ring. These amphipathic
molecules have been isolated from diverse natural sources,
including plants,[1] marine sources,[2] microorganisms,[3]
and humans.[4] In particular, mammalian sphingolipids have
various cellular functions, and play important roles in
membrane structure and cell signaling.[5]
Several analytical methods have been used to study
glycosphingolipids extracted from biological samples.[6,7]
Mass spectrometry is an important procedure for structural
studies, and the structures of sphingolipids have been
characterized using various ionization methods, including
electron ionization (EI),[8,9] electrospray ionization (ESI),[10,11]
and fast atom bombardment (FAB).[12–14] Recently, ESI
tandem mass spectrometry (MS/MS) connected to liquid
chromatography has been used to isolate and identify
mixtures of sphingolipids, and low-energy collision-induced
dissociation (CID) MS/MS has also been used in structural
analyses.[15–17] Over the past decades, FAB-MS/MS has
been used to characterize various sphingolipids because
it offers many advantages for structural elucidation and has
high-energy CID capabilities.[13,18–20] In our previous work,
FAB-MS/MS with a four-sector instrument was shown to be a
In the present study, the structures of six cerebrosides
(AA-1 to AA-6; Fig. 1) isolated from starfish egg extracts were
determined using high-energy CID tandem mass analyses
with a four-sector tandem mass spectrometer. Additionally,
double-bond configurations and sugar structures were
1
determined using H, 13C NMR and chemical degradation.
In this paper, we report on the detailed structural elucidation
of cerebrosides isolated from starfish eggs.
EXPERIMENTAL
Extraction and isolation
Raw starfish eggs (4.34 kg) were extracted with methanol
at room temperature. The crude extract was partitioned
between butanol and water, and the butanol layer was eluted
using silica gel column chromatography based on solvent
polarity. Neutral lipid fractions were eluted with acetone,
* Correspondence to: Y. H. Kim, Division of Mass Spectrometry
Research, Korea Basic Science Institute, 804-1 Yangcheong-ri,
Ochang-eup, Cheongwon-gun, Chungbuk 863-883, Korea.
E-mail: yhkim@kbsi.re.kr
Rapid Commun. Mass Spectrom. 2011, 25, 572–578
Copyright ß 2011 John Wiley & Sons, Ltd.

Characterization of cerebrosides isolated from starfish A. amurensis
O
16'
O
HN
OH
O
16'
OH
4
AA-2
HN
OH
O
HO
HO
O
22
AA-1
OH
9
4
OH
HO
HO
O
22
OH
O
13
OH
OH
O
18'
15'
HN
OH
O
24''
HN
OH
AA-4
OH
O
18
8
10
4
HO
HO
O
OH
AA-3
8
10
4
HO
HO
O
18
OH
OH
OH
OH
O
O
15'
24''
HN
23'
OH
HN
14'
OH
O
OH
8
10
OH
O
4
AA-6
HO
HO
AA-5
18
O
8
10
18
4
HO
HO
O
OH
OH
OH
OH
Figure 1. Structures of six cerebrosides isolated from A. amurensis starfish eggs.
and the major component was identified as cerebrosides. The
acetone fraction (500 mg) was applied to reversed-phase
high-performance liquid chromatography (HPLC) on a
YMC-ODS column (250 ꢀ 10 mm i.d.; YMC Co., Kyoto,
Japan), eluting with 100% methanol to yield six cerebrosides:
AA-1 (16 mg), AA-2 (12 mg), AA-3 (10 mg), AA-4 (20 mg),
AA-5 (10 mg), and AA-6 (15 mg).
RESULTS AND DISCUSSION
FAB of cerebrosides AA-1 to AA-6 isolated from starfish eggs
using 3-nitrobenzyl alcohol (3-NBA) saturated with sodium
iodide (NaI) yielded prominent sodium-adducted molecules
([M þ Na]þ) with peaks at m/z 792.6, 792.6, 858.6, 776.6, 830.6,
and 844.6 in each mass spectrum. The FAB mass spectrum
and results for AA-1 are shown in Fig. 2(a) and listed in
Table 1, respectively. The molecular formula of AA-1 was
determined as C44H83O9NNa using high-resolution mass
measurements at m/z 792.5968 (D þ0.3 mmu). This result is
summarized in Table 1 with the results of other cerebrosides.
In the FAB mass spectrum of AA-1, abundant fragment ions
corresponding to T and O ions identified by Ann and
Adams[19] were observed at m/z 320 and 538, respectively.
High-energy CID of the sodium- adducted molecules
fragmented extensively and provided information about
the polar head group, the composition of sphingoid bases and
the fatty acid portion from the presence of diagnostic ions
(Fig. 2(b)). The product ions (1,5X and 0,2X), generated by
cross-ring cleavage of the sugar ring, were observed at m/z
658 and 672, and B, C, and Y ions, from glycosidic bond
cleavage between the sugar ring and the sphingoid long-
chain base, were observed at m/z 185, 203, and 630,
respectively. Our notation follows the common nomenclature
for carbohydrate fragmentations proposed by Domon and
Costello.[18] These structural features indicated that the sugar
residue was the polar head group. The concomitant cleavage
of the glycosidic O–C bond and the C–C bond between the
3-hydroxy carbon of LCB and the adjacent carbon formed
prominent T ions at m/z 320, providing information on the
composition (h16:0) of the fatty acyl group. The predominant
O ion at m/z 538 arose from cleavage of the amide N–CO
bond with hydrogen transfer of an a-hydroxy group on the
N-acyl chain, providing information about the composition
(d22:2) of the sphingoid LCB. Moreover, there were two
abundant series of high-mass product ions, formed by
charge-remote cleavage of the fatty acyl chain and the long
chain of the sphingoid base (Fig. 3(a)). A series of homologous
ions generated from CnH2nþ2 losses along the saturated fatty
acyl chain via charge-remote elimination reactions[25]
appeared from m/z 776 to 594 with a 14 Da interval, with
the exception of a 16 Da mass difference, due to CH4 loss from
Acidic methanolysis
Each cerebroside (3 mg) was heated with 3 mL of 1 N HCl in
80% methanol at 708C overnight in a small sealed vial. They
were methanolized to yield a mixture of fatty acid methyl
esters (FAME), free sphingoid long-chain bases (LCB),
and methyl-D-glucopyranoside. The reaction mixture was
evaporated and partitioned with n-hexane and methanol. The
hexane and methanol layers were concentrated to produce
the FAME and free sphingoid LCB, respectively.
Mass spectrometry
All mass spectrometric analyses were performed using a
four-sector tandem mass spectrometer (JMS-HX110/110A,
JEOL). The ion source was operated at an accelerating voltage
of 10 kV in positive-ion mode. Ions were produced by FAB
using a cesium ion beam generated from the ion gun and
accelerated to 22 kV. Samples were dissolved in chloroform/
methanol (2:1, v/v). An aliquot of 1 mL of the solution was
mixed with 1 mL of 3-nitrobenzyl alcohol (3-NBA) saturated
with NaI on the FAB probe tip. CID of the precursor ions
selected by first mass spectrometer (E1B1) occurred in the
collision cell located between B1 and E2 and floated at 3.0 kV.
The product ions were analyzed using a B/E scanning
method with a second mass spectrometer (E2B2). Helium was
introduced into the collision chamber as a collision gas at a
pressure sufficient to reduce the precursor ion signal by 70%.
NMR
1H and 13C NMR spectra were recorded on a Varian UNITY
500 NMR spectrometer working at 500 MHz for proton and
1
125 MHz for carbon. The H and 13C NMR chemical shifts
were attributed to CD3OD at 3.30 and 49.0 ppm.
Rapid Commun. Mass Spectrom. 2011, 25, 572–578
Copyright ß 2011 John Wiley & Sons, Ltd.
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T. Park et al.
Figure 2. (a) Mass spectrum of AA-1 ionized by FAB after mixing with a 3-NBA matrix saturated with NaI and (b) high-energy
CID tandem mass spectrum of the sodium-adducted molecule, [M þ Na]þ, of AA-1 observed at m/z 792.6. Fragmentation
pathways observed in the CID of [M þ Na]þ are also shown. All product ions contained sodium.
the alkyl end. In the fragmentation study of ceramides by
Ann and Adams,[13] the abundant peaks at m/z 594 and 608
corresponded to J and K ions, respectively, and provided
information on the composition of the N-acyl chain. The
N-fatty acyl group of the AA-1 cerebroside was identified as a
2-hydroxy-N- palmitoyl group. The spectral pattern of these
homologous ions was similar to that of methyl
2-hydroxylhexadecanoate, synthesized by acidic methanoly-
sis of the cerebroside (Fig. 3(b)). The abundance of peaks
observed at m/z 594 (J ion) and 111 in Figs. 3(a) and 3(b),
respectively, was due to the conjugation stability of the
product ion, N-acyl aldehyde, which formed via the cleavage
of the C–C bond adjacent to an a-hydroxyl group and
concomitant hydrogen transfer.
The concurrent observation of an even-electron ion at m/z
111 and an odd-electron radical ion at m/z 112 in the
high-energy CID spectrum of FAME demonstrated that the
charge-remote fragmentation (CRF) along the long hydro-
carbon chain occurred in two steps: homolytic cleavage of a
C–C bond, followed by elimination of a radical (usually Hꢁ)
Table 1. Summary of cerebroside species isolated from Asterias amurensis starfish eggs
T, O ions,
[M þ Na]þ, m/z
m/za
Dm/M
Species
Observed
Theoretical
(ppm)
Molecular formula
T
O
Sphingoid base/N-acyl groupb
AA-1
AA-2
AA-3
AA-4
AA-5
AA-6
792.5968
792.5966
858.6433
776.5658
830.6124
844.6279
792.5966
792.5966
858.6435
776.5653
830.6122
844.6279
0.3
0
–0.2
0.7
0.2
0.1
C44H83O9NNa
C44H83O9NNa
C49H89O9NNa
C43H79O9NNa
C47H85O9NNa
C48H87O9NNa
320
320
430
348
416
430
538
538
494
494
480
480
d22:2D4,13/h16:0
d22:2D4,9/h16:0
d19:3 9-methyl-D4,8,10/h24:1D15’
d19:3 9-methyl-D4,8,10/h18:0
d18:3 D4,8,10/h23:1D14’
d18:3 D4,8,10/h24:1D15’
a T and O ions provided information about the compositions of N-fatty acyl groups and sphingoid bases, respectively.
b The numbers after D indicate the position of the double bonds present in sphingoid bases and fatty acyl chains. The
superscript 9-methyl indicates that the methyl group was positioned at C-9 of the sphingoid chain.
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Characterization of cerebrosides isolated from starfish A. amurensis
Figure 3. Comparison of the peak patterns from charge-remote fragmentation along the hydrocarbon chains of AA-1 and its
fatty acid methyl ester (FAME) and sphingosine derived by acidic methanolysis. (a) High-mass region of the tandem mass
spectrum shown in Fig. 2(b) and high-energy CID tandem mass spectra of sodium-adducted molecules, [M þ Na]þ, of (b) FAME
and (c) sphingosine. The pathways of the homologous ions generated by CRFs along the hydrocarbon chains of each [M þ Na]þ
ion are also shown, along with their structures. In (a), each series of ions from CRFs along the sphingoid chain and the N-acyl
group are labeled in blue and red, respectively, and the common ions are labeled in black. An equals sign above a peak indicates
the double-bond position.
and thermal-like 1,4-eliminations.[26] The CRF along the LCB
chain resulted in a series of product ions with the neighboring
peaks in the series separated by 14 Da, including common
ions of two series from m/z 776 to 678. However, the presence
of double bonds in the LCB chain reduced the neighboring
peak separation to 12 Da, similar to the result from our
previous high-energy CID studies of glycoglycerolipids.[21]
The spectral pattern in the LCB series at m/z 666(¼), (where
’ ¼ ’ represents the double-bond position), 652, 638, 624,
610, 596, 582, 568, 554, 542(¼), 528, and 498 (G ion, denoted
according to Ann and Adams[13]) identified the positions of
double bonds and hydroxyl group. Thus, the sphingosine
unit of AA-1 was characterized as docosasphinga-(4,13)-
dienine. The similarity of the spectral patterns between this
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T. Park et al.
Figure 4. High-mass region of tandem mass spectra for the [M þ Na]þ ions of (a) AA-2, (b) AA-3, and (c) AA-4. Each series of
ions from CRFs along the sphingoid chain and the N-acyl group are labeled in blue and red, respectively, and the common ions
are labeled in black. An equal sign above a peak indicates the double-bond position.
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Characterization of cerebrosides isolated from starfish A. amurensis
series of ions and the corresponding ions of sphingosine
obtained by methanolysis of AA-1 confirmed the structure of
the sphingosine unit (with the exception of odd-electron ions
at m/z 83, 153, and 277). In particular, the abundance of the
peaks at m/z 498, 582, 638, and 706 and the weakness of the
peaks at m/z 528, 542(¼), 554, and 568 (Fig. 3(a)) corresponded
to those of the ions at m/z 82, 166, 222, and 290 and the ions at
m/z 112, 126(¼), 138, and 152 (Fig. 3(c)), respectively. The
odd-electron ions in Fig. 3(c), formed via homolytic cleavage
of C–C bonds, indicated abundant peaks because the allylic
radical was stabilized by a double bond. Thus, in addition to
the mass difference between neighboring ions, relative peak
intensities were also helpful in the determination of the
double-bond positions in the N-acyl chain and the sphingoid
LCB.
the AA-1 cerebroside was determined to be (4E,13Z)-1-
(b-D-glucopyranosyloxy)-3-hydroxy-2-[2-hydroxyhexadeca-
noyl]amino-4,13-docosadiene. The high-energy CID spec-
trum (Fig. 4(a)) of sodium-adducted molecules, m/z 792.6 of
AA-2, was analogous to that of AA-1, except for a spectral
difference due to the double bonds positioned at C-4 and C-9
on the sphingoid base. The gross structure of the AA-2
cerebroside was characterized as (4E,9Z)-1-(b-D-gluco-
pyranosyloxy)-3-hydroxy-2-[2-hydroxyhexadecanoyl]amino-
4,9-docosadiene. The T and O ions in the high-energy CID
spectrum of the sodium-adducted molecules, m/z 858.6 of
AA-3, were observed at m/z 430 and 494, respectively
(Fig. 4(b)). The compositions of the N-acyl chain and the
sphingoid LCB from these ions were assigned as h24:1 and
d19:3, respectively. The product ion peak at m/z 732 from the
series of ions generated by the fragments of the N-acyl chain,
starting from the J ion at m/z 550, had a mass difference of
12 Da relative to the neighboring ion at m/z 744. The
double-bond position at C-15’ was confirmed by the
1
On the other hand, the H and 13C NMR study of AA-1
revealed that the sugar moiety was a b-glucopyranoside,
and D4 and D13 double bonds in the sphingoid LCB had E
and Z geometries, respectively. Thus, the gross structure of
Figure 5. High-mass region of tandem mass spectra for the [M þ Na]þ ions of (a) AA-5 and (b) AA-6. The peak intensity in each
MS/MS spectrum was normalized to that of the most abundant O ion.
Rapid Commun. Mass Spectrom. 2011, 25, 572–578
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T. Park et al.
abundant peak at m/z 704, due to the cleavage of the allylic
C–C bond. On the other hand, the positions of double bonds
in the LCB chain were at C-4, C-8, and C-10, according to the
spectral pattern of the series of ions appearing at m/z 608
(G ion), 638, 652(¼), 664, 678, 692, 706(¼), 732, and 746(¼),
including common ions of two series from m/z 842 to 758.
However, the methyl group was expected to be at C-9 or C-10
due to the presence of ions at m/z 718 and 732, which
commonly occur with CRF ions of the N-acyl group. The peak
intensity at m/z 732 was higher than that at m/z 718 because of
the peak that occurred with the fragmentation of the N-acyl
group. The high-energy CID spectrum (Fig. 4(c)) of AA-4,
with the same sphingoid composition but the saturated
N-acyl group (h18:0), showed that the peaks at m/z 624(¼),
650, and 664(¼) were not common to those of the saturated
N-acyl group. The comparison demonstrated that the
methyl group on the sphingoid LCB was positioned at C-9
in the middle of the (4,8,10)-sphingenine residue. Based on
the these results and NMR data, AA-3 and AA-4 were
characterized as (4E,8E,10E)-1-(b-D-glucopyranosyloxy)-3-
hydroxy-2-[2-hydroxy-15-tetracosenoyl]amino-9-methyl-4,8,10-
octadecatriene and (4E,8E,10E)-1-(b-D-glucopyranosyloxy)-
3-hydroxy-2-[2-hydroxyoctadecanoyl]amino-9-methyl-4,8,10-
octadecatriene, respectively.
rapid method has great potential for determining the
complete structure of individual components present in a
mixture of sphingolipids isolated from biological sources,
including sphingosine, ceramides, and sphingomyelins.
Acknowledgements
This work was supported by Grant No. G30123 from the
Korea Basic Science Institute.
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