Báo cáo khoa học: Repeats of LacdiNAc and fucosylated LacdiNAc on N-glycans of the human parasite Schistosoma mansoni doc

Repeats of LacdiNAc and fucosylated LacdiNAc on
N-glycans of the human parasite Schistosoma mansoni
Manfred Wuhrer, Carolien A. M. Koeleman, Andre
´
M. Deelder and Cornelis H. Hokke
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center,
The Netherlands
The most common complex-type N-glycans of mam-
mals contain N-acetyllactosamine-type antennae [Lac-
NAc; Gal(b1–4)GlcNAc(b1-], which can be substituted
in various positions by fucoses, sialic acids, sulfate,
glucuronic acid, etc. LacNAc-type antennae are also
seen in other eukaryotes [1–5]. The arising terminal
structures can be antigenic [2,6] or may act as ligands
of lectins [7,8]. They may modify cell–cell interaction,
for example in development [9–12] and cancer [13–15].
An alternative antennary structure is N,N¢-diacetyl-
lactosediamine [LacdiNAc or LDN; GalNAc(b1–
4)GlcNAc(b1-], which may likewise be modified by
various substituents such as fucose, sulfate or sialic
acid. LDN-based motifs have been found on N- or
O-glycans of various mammalian glycoproteins inclu-
ding seminal plasma glycodelin [16], thyrotropin [17]
and tissue factor pathway inhibitor [18], but are also
expressed by various pathogens, including the human
parasite Schistosoma mansoni [2]. Glycans with LDN
and LDNF {GalNAc(b1–4)[Fuc(a1–3)]GlcNAc(b1-}
expressed by S. mansoni are targets of the humoral
immune responses of the host [19], and LDN-contain-
ing glycoconjugates of schistosomes may be ligands for
galectin-3-mediated immune recognition [7].

In humans, biosynthesis of LDN can occur by
two different b1–4-N-acetylgalactosaminyltransferases:
b4GalNAc-T3, which is expressed in stomach, colon
and testes at high levels [20]; and b4GalNAc-T4, which
is transcribed in ovaries and brain tissues [21]. From
the nematode Caenorhabditis elegans, a b1–4-galactos-
aminyltransferase involved in LDN has likewise been
identified [22]. The expression of this enzyme in Chi-
nese hamster ovary (CHO) Lec8 cells has led to the
production of N-glycans with LDN repeats, which
Keywords
mass spectrometry; parasite; terminal
N-acetylgalactosamine; trematode
Correspondence
M. Wuhrer, Department of Parasitology,
Leiden University Medical Center, P.O. Box
9600, 2300 RC Leiden, the Netherlands
Fax: +31 71 526 6907
Tel: +31 71 526 5077
E-mail:
(Received 15 September 2005, revised
11 November 2005, accepted 21 November
2005)
doi:10.1111/j.1742-4658.2005.05068.x
N-Glycans from glycoproteins of the worm stage of the human parasite
Schistosoma mansoni were enzymatically released, fluorescently labelled and
analysed using various mass spectrometric and chromatographic methods.
A family of 28 mainly core-a1–6-fucosylated, diantennary N-glycans of
composition Hex
3)4

HexNAc
6)12
Fuc
1)6
was found to carry dimers of N,N¢-
diacetyllactosediamine [LacdiNAc or LDN; GalNAc(b1–4)GlcNAc(b1-]
with or without fucose a1–3-linked to the N-acetylglucosamine residues
in the antennae {GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1–3)GalNAc(b1–
4)[±Fuc(a1–3)]GlcNAc(b1-}. To date, oligomeric LDN and oligomeric
fucosylated LDN (LDNF) have been found only on N-glycans from
mammalian cells engineered to express Caenorhabditis elegans b4-GalNAc
transferase and human a3-fucosyltransferase IX [Z. S. Kawar et al. (2005)
J Biol Chem 280, 12810–12819]. It now appears that LDN(F) repeats can
also occur in a natural system such as the schistosome parasite. Like
monomeric LDN and LDNF, the dimeric LDN(F) moieties found here are
expected to be targets of humoral and cellular immune responses during
schistosome infection.
Abbreviations
2AB, 2-aminobenzamide; CHO, Chinese hamster ovary cells; F, deoxyhexose; H, hexose; IT, ion-trap; LacNAc, Gal(b1–4)GlcNAcb1-; LC,
liquid chromatography; LDN or LacdiNAc, N,N¢-diacetyllactosediamine; LDNF, GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1-; N, N-acetylhexosamine;
PNGase F, peptide N-glycosidase F.
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 347
were converted to LDNF repeats by coexpression of
human a1–3-fucosyltransferase IX [23]. The mam-
malian b1–3-N-acetylglucosaminyltransferase(s) which
contribute(s) to the synthesis of this alternating chain
have yet to be identified [23].
Whereas LDN repeats have thus been registered
after expressing a b4-GalNAc transferase in a hetero-
logous system, oligo- or poly LDN units have hitherto

not been described for natural sources. Here, we des-
cribe the expression of dimeric, in part fucosylated
LDN {GalNAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1–3)Gal-
NAc(b1–4)[±Fuc(a1–3)]GlcNAc(b1-} on N-glycans of
S. mansoni adults.
Results
Evidence for N-glycans with chains of four
HexNAc residues
N-Glycans were released from a total (glyco)protein
preparation of S. mansoni adult worms using peptide
N-glycosidase F (PNGase F) treatment and analysed
by MALDI-TOF-MS (Fig. 1). Almost all groups of
glycans thus characterized had compositions in accord-
ance with the published structures of N-glycans from
S. mansoni adult worms [2–5] (Fig. 1). Glycans of com-
position H
2)10
N
2
were interpreted as being oligoman-
nosidic structures with an additional terminal glucose
residue for the H
10
N
2
species. H
2)4
N
2
F

1
represents
a group of core-a1–6-fucosylated, paucimannosidic
N-glycans. H
3
N
4
F
0)2
and H
3
N
6
F
1)3
are in agreement
with complex-type structures containing one or two
LDN or fucosylated LDN (GalNAc(b1–4)[Fuc(a1–
3)]GlcNAc(b1-; LDNF) antennae. Glycans with com-
positions of H
4
N
6
F
1
,H
4
N
5
F

0)3
,H
5
N
6
F
1)4
,H
4
N
4
F
1)2
,
H
5
N
5
F
0)2
and H
3
N
5
F
1
were interpreted as containing
two or more LDN and ⁄ or partially truncated LacNAc
antennae. H
4

N
3
F
1)2
,H
5
N
4
F
0)3
,H
6
N
5
F
0)4
,H
7
N
6
F
1
most likely correspond to hybrid-type structures with
1, 2, 3 or 4 LacNAc and ⁄ or Lewis X antennae,
although there are no detailed data in the literature to
substantiate this.
A
B
Fig. 1. MALDI-TOF-MS of N-glycans released from S. mansoni adult worms. (A) Low mass range; (B) high mass range; ·8, intensities eight
times enlarged; F, deoxyhexose; H, hexose; N, N-acetylhexosamine. N-glycan species containing eight or more HexNAc residues are labelled

in bold-type.
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
348 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
One particular group of glycans, however, which
showed compositions of H
3
N
8)12
F
1)5
(Fig. 1), attrac-
ted our attention by its high N-acetylhexosamine con-
tent which led to the hypothesis that they contain
elongated N-acetylhexosamine stretches on their anten-
nae.
To test this hypothesis, RP-nano-LC-ESI-IT-MS ⁄
MS was performed on the N-glycans of S. mansoni
adult worms after labelling with 2-aminobenzamide
(2AB; Fig. 2). MS ⁄ MS data, obtained in the automatic
mode, were screened for fragment ions that indicate
oligo-N-acetylhexosamine stretches. A fragment ion
corresponding to three N-acetylhexosamine residues
(m ⁄ z 632) was found for the precursor at m ⁄ z 1017
([M + 2Na]
2+
) corresponding to the N-glycan
H
3
N
6

F
1
A(A¼ 2-aminobenzamide). The fragment ion
spectrum (Fig. 2B) indicated that at least part of the
N-glycans of this composition contained N-acetylhexo-
samine chains of three or more residues.
Because the MALDI-TOF-MS profile indicated that
many of the N-glycans with high N-acetylhexosamine
content were of low abundance, we chose a two-
dimensional HPLC approach to allow MS ⁄ MS analy-
sis of most of the species. In the first dimension, the
2AB-labelled N-glycans were separated by normal-
phase HPLC on an amide column (Fig. 3). Individual
peak fractions were analysed by MALDI-TOF-MS
and separated in the second dimension by RP-nano-
LC-IT-MS with automatic acquisition of MS ⁄ MS
data. In order to obtain extensive MS ⁄ MS data of
both sodium and proton adducts, each peak fraction
was analysed by nano-LC-MS both with and with-
out addition of sodium hydroxide to the running
solvents, resulting in the registration ⁄ fragmentation of
0
2
612
1
8
)ni
m
(emiT
CPB

71
01C
I
E
236)SM/SM(CIE
Intensity
A
364.2 N
1
A
429.2 N
2
510.2 N
1
F
1
A
632.3 N
3
713.4 N
2
F
1
A
741.9
814.4
842.9
906.9
944.4
1017.5

1118.4 H
3
N
3
1199.4 H
3
N
2
F
1
A
1256.4
1321.4 H
3
N
4
1402.5 H
3
N
3
F
1
A
1460.5
1524.5 H
3
N
5
1605.5 H
3

N
4
F
1
A
00
4100210001008006004 0
061
z/
m
916.1
1008.5
Intensity
H
3
N
6
F
1
A
#
SM
2
H)7101(
3
N
6
F
1
A

B
Fig. 2. RP-nano-LC-MS ⁄ MS of 2-aminobenzamide-labelled N-glycans from adult S. mansoni. (A) Chromatogram indicating the presence of
H
3
N
6
F
1
A N-glycan species (double sodiated; EIC 1017) leading to a fragment ion at m ⁄ z 632 which corresponds to sodiated N
3
. (B) Fragment
ion spectrum of the H
3
N
6
F
1
A N-glycan species. A, 2-aminobenzamide; BPC, base peak chromatogram; EIC, extracted ion chromatogram;
F, deoxyhexose; H, hexose; N, N-acetylhexosamine; double-headed arrow with continuous line, N-acetylhexosamine; double headed arrow
with dashed line, deoxyhexose; #, double-sodiated species.
M. Wuhrer et al. S. mansoni N-glycans with dimeric LacdiNAc
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 349
predominantly sodium adducts or proton adducts,
respectively.
For glycans of composition H
3
N
6
F
1

A, which con-
tain, at least in part, N-acetylhexosamine stretches
(Fig. 2), this two-dimensional HPLC approach
resolved three isomers eluting in normal-phase frac-
tions 24 and 26 (Fig. 4). The MS ⁄ MS data obtained
for these isobaric N-glycans (Fig. 5) indicated that iso-
mers 1 and 2 were diantennary N-glycans with two
LDN antennae differing only in the fucose attachment
site, i.e. core-(a1–6)-fucosylation for isomer 1 and
antenna-fucosylation for isomer 2 (Figs 4 and 5). Iso-
mer 3, however, exhibited a stretch of four N-acetyl-
hexosamine residues, which was tentatively interpreted
as a diLDN unit [GalNAc(b1–4)GlcNAc(b1–3)Gal-
NAc(b1–4)GlcNAc(b1-].
Overall screening of the two-dimensional LC-MS ⁄
MS data set revealed a large group of N-glycans exhib-
iting fragment ions indicative of HexNAc chains, as
summarized in Table 1. For many of the N-glycan spe-
cies, MS ⁄ MS data were obtained for sodium adducts
Fig. 3. Normal-phase HPLC separation of 2-aminobenzamide-labelled N-glycans from adult S. mansoni. Peaks are labelled with fraction num-
bers and glycan compositions of the major 2-aminobenzamide-labelled species detected. Retention times of 2-aminobenzamide-labelled par-
tial hydrolysate of dextran are indicated in italics in the upper part of the figure. All fractions were subjected to RP-nano-LC-MS ⁄ MS.
Fractions containing species with a fragment ion at m ⁄ z 632 corresponding to sodiated N
3
and ⁄ or an ion at m ⁄ z 813 corresponding to proto-
nated N
4
are labelled with (+). An overview of the N-glycan species giving rise to these characteristic ions is given in Table 1. F, deoxy-
hexose; H, hexose; N, N-acetylhexosamine.
0

25
1
42.rF
1
62.rF
3
2
Intensity
)
nim(emiT
A
A
A
Fig. 4. RP-nano-LC-MS ⁄ MS of normal-phase HPLC fractions 24 and
26. Extracted ion chromatograms of m ⁄ z 1017 displayed for frac-
tions 24 and 26 indicated three isomers separated by the two-
dimensional HPLC system. The assigned structures are based on
the MS ⁄ MS spectra shown on Fig. 5. Yellow square, N-acetylgal-
actosamine; blue square, N-acetylglucosamine; green circle, man-
nose; red triangle, fucose; A, 2-aminobenzamide.
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
350 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
as well as proton adducts, with both fragment ion
spectra revealing valuable structural information inclu-
ding the HexNAc chain lengths (exemplified in Fig. 6).
In the case of the protonated species, the length of the
HexNAc chain was clearly indicated by the intense
protonated HexNAc
4
(m ⁄ z 813), whereas the protonat-

ed HexNAc
3
fragment was less abundant (m ⁄ z 610;
Fig. 6A), indicating a particular lability of the Hex-
NAc-Hex linkage. Although the fragment ion spectrum
of the sodiated species likewise exhibited HexNAc
3
and HexNAc
4
ions (m ⁄ z 632 and 835), the intensity
ratios were the reversed. The innermost linkage of the
antenna HexNAc chain appeared to be particularly
labile, resulting in an intense HexNAc
3
peak and
a low-abundance signal for the HexNAc
4
sodium
adduct (Fig. 6B). For some species, at least part of the
HexNAc chains seemed to be fucosylated as indicated
by the corresponding reporter ions (e.g. m ⁄ z 778 for
sodiated N
3
F
1
). In all cases the length of the HexNAc
chain was deduced to be four HexNAc residues, and
none of the fragment ion spectra indicated species with
HexNAc chains of three or five HexNAc residues.
Detailed characterization of N-glycans

Few of the identified N-glycans with chains of four
HexNAc were obtained in sufficient quantities and
purity to allow more detailed structural characteriza-
tion: the glycan species H
3
N
8
F
4
A detected in fraction
39 (Table 1) was purified by preparative RP-HPLC
(not shown) and subjected to permethylation. Frag-
ment ion analysis of permethylated H
3
N
8
F
4
A using
346.1
429.1
510.2
590.2
639.6
#
713.2
#
741.6
#
814.3

#
843.0
#
916.3
#
944.3
#
1008.9
#
1053.2
1118.3
1210.3
1256.3
1321.4
1402.4
1459.5
1524.4
1605.5
1697.9
632.2
364.3
429.1
510.2
567.1
671.4
713.1
#
842.7
#
916.2

#
944.3
#
1008.9
#
1053.5
1118.3
1200.4
1256.4
1321.3
1344.2
1402.5
1460.5
1524.5
1605.5
1649.3
004
0
06
008 0
00
1 0021 0041
0
061
A
A
A
364.1
A
A

A
A
1199.3
1587.4
A
A
A
835.3
#
A
A
814.6
#
A
1remosi
364.1
429.1
567.2
639.7
#
741.2
#
814.8
#
842.8
#
916.3
#
944.3
#

1008.4
#
1053.3
1118.4
1256.5
1459.4
1524.5
1605.5
A
A
A
A
A
B 2remosi
C 3remosi
A
Intensity
z/
m
Fig. 5. Fragment ion spectra (nano-LC-ESI-IT-MS ⁄ MS) of the three isomers displayed in Fig. 4. The deduced structures are boxed. Yellow
square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; double-
headed arrow with continuous line, N-acetylhexosamine; double-headed arrow with dashed line, deoxyhexose; #, double-sodiated fragment.
M. Wuhrer et al. S. mansoni N-glycans with dimeric LacdiNAc
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 351
Table 1. Analytical data for S. mansoni adult worm N-glycans exhibiting partially fucosylated diLDN antennae. Compositions are given in
terms of hexose (H), N-acetylhexosamine (N), and deoxyhexose (fucose; F). Fragment ions were determined by nano-LC-ESI-IT-MS ⁄ MS
selecting sodiated and ⁄ or protonated precursors. n. d., not done.
Composition
HPLC
fractions

Ions registered by
nano-LC-ESI-IT-MS (m ⁄ z)
Characteristic
fragmentions (m ⁄ z) Proposed structural features
H
3
N
6
F
1
26 1893.1
b
[M + Na]
+
; 1017.4 [M +2Na]
2+
Fig. 5C core-(a1–6)-fucosylation, LDN-LDN- antenna
H
3
N
6
F
2
37 2038.9
b
[M + Na]
+
; 1068.6 [M +2H]
2+
407 (N

2
)
a
, 553 (N
2
F
1
)
a
,
813 (N
4
)
a
, 778 (N
3
F
1
)
no core-fucosylation, LDNF-LDNF- antenna
H
3
N
6
F
3
39 2185.2
b
[M + Na]
+

; 1141.6 [M +2H]
2+
;
1163.4 [M +2Na]
2+
510.2 (N
1
F
1
A), 778 (N
3
F
1
),
553 (N
2
F
1
)
a
, 610 (N
3
)
a
,
699 (N
2
F
2
)

a
, 756 (N
3
F
1
)
a
,
813 (N
4
)
a
, 959 (N
4
F
1
)
a
,
1106 (N
4
F
2
)
a
core-(a1–6)- fucosylation, LDNF-LDNF- antenna
H
3
N
7

F
1
27 2096.1
b
[M + Na]
+
; 1196.9 [M +2H]
2+
;
1218.9 [M +2Na]
2+
488.1 (N
1
F
1
A)
a
, 813 (N
4
)
a
,
632 (N
3
), 835 (N
4
),
1218.4 (H
2
N

3
F
1
A)
a
core-(a1–6)- fucosylation, LDN- LDN- antenna,
1 antenna of a single HexNAc residue
(truncated)
H
4
N
7
F
4
40 2696.0
b
[M + Na]
+
; 932.1 [M +3H]
3+
407 (N
2
)
a
, 512 (H
1
N
1
F
1

)
a
,
813 (N
4
)
a
, 959 (N
4
F
1
)
a
core-(a1–6)- fucosylation, 1 LDNF-LDNF- antenna,
1 Lewis X- antenna
H
3
N
8
F
1
30 2299.0
b
[M + Na]
+
; 1198.5 [M +2H]
2+
;
1220.5 [M +2Na]
2+

Fig. 6 core-(a1–6)- fucosylation, LDN- LDN- antenna,
LDN- antenna
H
3
N
8
F
2
37 2445.1
b
[M + Na]
+
; 1271.5 [M +2H]
2+
;
1293.5 [M +2Na]
2+
n.d. 1 LDN-LDN- antenna, 1 LDN- antenna,
both possibly fucosylated
H
3
N
8
F
3
37 2591.6
b
[M + Na]
+
; 896.9 [M +3H]

3+
;
918.8 [M +3Na]
3+
510 (N
1
F
1
A), 575 (N
2
F
1
),
778 (N
3
F
1
),
989 [M +2Na]
2+
(H
3
N
5
F
2
A),
1549 (H
3
N

3
F
2
A)
core-(a1–6)- fucosylation, 1 LDNF- LDNF- antenna,
1 LDN- antenna
H
3
N
8
F
4
38–40 2737.5
b
[M + Na]
+
; 945.6 [M +3H]
3+
;
967.4 [M +3Na]
3+
; 1439.6 [M +2Na]
2+
496 (N
1
F
2
)
a
, 699 (N

2
F
2
)
a
,
813 (N
4
)
a
, 1106 (N
4
F
2
)
a
,
510 (N
1
F
1
A), 632 (N
3
),
778 (N
3
F
1
),
1062 [M +2Na]

2+
(H
3
N
5
F
3
A),
1164 [M +2Na]
2+
(H
3
N
6
F
3
A),
1338 [M +2Na]
2+
(H
3
N
7
F
4
A),
1810 (H
3
N
4

F
2
A),
1900 (H
3
N
4
F
3
A);
see also Fig. 7
core-(a1–6)- fucosylation, 1 LDNF- LDNF- antenna,
1 LDNF- antenna
H
3
N
9
F
1
37 2502.2
b
[M + Na]
+
; 1300.0 [M +2H]
2+
;
1321.9 [M +2Na]
2+
n.d. 1 LDN-LDN- antenna, 1 LDN- antenna,
1 antenna of a single HexNAc residue (truncated)

H
3
N
9
F
2
37 2648.4
b
[M + Na]
+
; 916.0 [M +3H]
3+
;
937.7 [M +3Na]
3+
813 (N
4
)
a
, 959 (N
4
F
1
)
a
,
510 (N
1
F
1

A), 778 (N
3
F
1
)
core-(a1–6)- fucosylation, 1 LDNF-LDN- antenna,
1 antenna of a single HexNAc residue (truncated)
H
3
N
9
F
3
39 2795.0
b
[M + Na]
+
; 964.6 [M +3H]
3+
;
986.4 [M +3Na]
3+
510 (N
1
F
1
A), 699 (N
2
F
2

)
a
,
813 (N
4
)
a
, 575 (N
2
F
1
),
632 (N
3
), 778 (N
3
F
1
),
981 (N
4
F
1
),
1090 [M +2Na]
2+
(H
3
N
6

F
2
A),
1810 (H
3
N
5
F
1
A)
core-(a1–6)-fucosylation, 1 LDNF-LDNF- antenna,
1 antenna of a single HexNAc residue (truncated)
H
3
N
10
F
1
38 2705.7
b
[M + Na]
+
; 1423.5 [M +2Na]
2+
; n.d. 2 LDN-LDN antennae, possibly fucosylated
H
3
N
10
F

2
38 2851.5
b
; 1005.4 [M +3Na]
3+
510.2 (N
1
F
1
A), 632 (N
3
),
778 (N
3
F
1
), 981 (N
4
F
1
),
1192 [M +2Na]
2+
(H
3
N
7
F
1
A),

1753 (H
3
N
4
F
2
A),
1809 (H
3
N
5
F
1
A),
core-(a1–6)-fucosylation, 2 LDN-LDN- antennae,
one of them singly fucosylated
H
3
N
10
F
3
40 2996.5
b
[M + Na]
+
; 1032.3 [M +3H]
3+
488 (N
1

F
1
A)
a
, 813 (N
4
)
a
,
959 (N
4
F
1
)
a
core-(a1–6)-fucosylation, 2 LDN-LDN- antennae,
H
3
N
10
F
4
40–42 3143.9
b
[M + Na]
+
; 1081.2 [M +3H]
3+
;
1102.8 [M +3Na]

3+
510 (N
1
F
1
A), 813 (N
4
)
a
,
699 (N
2
F
2
)
a
, 1252 (N
4
F
3
)
a
,
575 (N
2
F
1
), 778 (N
3
F

1
),
1753 (H
3
N
4
F
2
A),
1899 (H
3
N
4
F
3
A)
core-(a1–6)-fucosylation, 1 LDNF-LDNF- antenna,
1 LDN-LDNF- antenna
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
352 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
Table 1. (Continued).
Composition
HPLC
fractions
Ions registered by
nano-LC-ESI-IT-MS (m ⁄ z)
Characteristic fragment
ions (m ⁄ z) Proposed structural features
H
3

N
10
F
5
42, 43 3289.5
b
[M + Na]
+
; 1129.8 [M +3H]
3+
;
1151.5 [M +3Na]
3+
813 (N
4
)
a
, 902 (N
3
F
2
)
a
,
1106 (N
4
F
2
)
a

,
510 (N
1
F
1
A), 575 (N
2
F
1
),
778 (N
3
F
1
), 1897 (H
3
N
4
F
3
A)
core-(a1–6)-fucosylation, 2 LDNF-LDNF-
antennae
H
3
N
11
F
1
37 2909.0

b
[M + Na]
+
; 1002.3 [M +3H]
3+
;
1024.4 [M +3Na]
3+
813 (N
4
)
a
; 1380 (H
3
N
3
F
1
A)
a
;
510 (N
1
F
1
A), 429 (N
2
),
632 (N
3

),
1119 [M +2Na]
2+
(H
3
N
8
F
1
A),
1221 [M +2Na]
2+
(H
3
N
9
F
1
A),
1322 [M +2Na]
2+
(H
3
N
10
F
1
A),
1809 (H
3

N
5
F
1
A), 1931 (H
3
N
7
)
core-(a1–6)-fucosylation, 2 LDN-LDN- antennae,
1 antenna of a single HexNAc residue
(truncated)
H
3
N
11
F
2
39, 40 1051.4 [M +3H]
3+
; 1072.8 [M +3Na]
3+
488 (N
1
F
1
A)
a
, 553 (N
2

)
a
,
813 (N
4
)
a
,
1170 [M +2H]
2+
(H
3
N
7
F
2
A)
a
,
1301 [M +2H]
2+
(H
3
N
9
F
1
A)
a
,

1374 [M +2H]
2+
(H
3
N
9
F
2
A)
a
,
1731 (H
3
N
4
F
2
A)
a
,
1294 [M +2Na]
2+
(H
3
N
8
F
2
A),
1090 [M +2Na]

2+
(H
3
N
6
F
2
A),
1323 [M +2Na]
2+
(H
3
N
9
F
1
A),
1810 (H
3
N
5
F
1
A),
1955 (H
3
N
5
F
2

A),
1752 (H
3
N
4
F
2
A)
core-(a1–6)-fucosylation,
2 partially fucosylated LDN-LDN- antennae,
1 antenna of a single HexNAc residue
(truncated) core-(a1–6)-fucosylation,
2 partially fucosylated LDN-LDN- antennae,
H
3
N
11
F
3
40, 41 1099.9 [M +3H]
3+
; 1121.8 [M +3Na]
3+
813 (N
4
)
a
, 959 (N
4
F

1
)
a
,
510 (N
1
F
1
A), 575 (N
2
F
1
),
778 (N
3
F
1
),
1293 [M +2Na]
2+
(H
3
N
8
F
2
A),
1954 (H
3
N

5
F
2
A)
1 antenna of a single HexNAc residue
(truncated) core-(a1–6)-fucosylation,
2 partly fucosylated LDN-LDN- antennae,
H
3
N
11
F
4
42, 43 1148.6 [M +3H]
3+
; 1170.5 [M +3Na]
3+
813 (N
4
)
a
, 699 (N
2
F
2
)
a
,
1252 (N
4

F
3
)
a
,510(N
1
F
1
A),
575 (N
2
F
1
), 778 (N
3
F
1
),
1955 (H
3
N
5
F
2
A)
1 antenna of a single HexNAc residue
(truncated)
H
3
N

11
F
5
43 1219.2 [M +3Na]
3+
510 (N
1
F
1
A), 429 (N
2
),
713 (N
2
F
1
A), 778 (N
3
F
1
),
2101 (H
3
N
5
F
3
A)
core-(a1–6)-fucosylation, 2 LDNF-LDNF-
antennae, 1 antenna of a single HexNAc

residue (truncated)
H
3
N
12
F
1
39 3111.8
b
[M + Na]
+
; 1092.1 [M +3Na]
3+
;n.d. –
H
3
N
12
F
2
40 3257.8
b
[M + Na]
+
; 1118.8 [M +3H]
3+
;
1140.8 [M +3Na]
3+
510 (N

1
F
1
A), 813 (N
4
)
a
,
959 (N
4
F
1
)
a
,
1120 [M +2H]
2+
(H
3
N
8
F
1
A)
a
,
1177 (H
3
N
2

F
1
A)
a
,
1273 [M +2H]
2+
(H
3
N
8
F
2
A)
a
,
1584 (H
3
N
4
F
1
A)
1
core-(a1–6)-fucosylation, 1 LDN-LDN- antenna,
1 LDNF-LDN- antenna, 1 LDN- antenna
H
3
N
12

F
3
41, 42 3403.7
b
[M + Na]
+
; 1167.6 [M +3H]
3+
;
1189.5 [M +3Na]
3+
510 (N
1
F
1
A), 575 (N
2
F
1
),
632 (N
3
),778 (N
3
F
1
),
2159 (H
3
N

6
F
2
A)
core-(a1–6)-fucosylation, 1 LDNF-LDN antenna,
1 LDN-LDN- antenna, 1 LDNF- antenna
H
3
N
12
F
4
43 1238.1 [M +3Na]
3+
510 (N
1
F
1
A), 575 (N
2
F
1
),
713 (N
2
F
1
A), 778 (N
3
F

1
),
2158 (H
3
N
6
F
2
A),
2304 (H
3
N
6
F
3
A)
core-(a1–6)-fucosylation, 2 partially fucosylated
LDN-LDN- antennae, 1 LDN(F)- antenna
H
3
N
12
F
5
43 1265.1 [M +3H]
3+
; 1286.9 [M +3Na]
3+
813 (N
4

)
a
, 699 (N
2
F
2
)
a
,
575 (N
2
F
1
), 778 (N
3
F
1
),
2304 (H
3
N
6
F
3
A)
no core-fucosylation, 2 LDNF-LDNF- antennae,
1 LDNF- antenna
H
3
N

12
F
6
43 1335.6 [M +3Na]
3+
510 (N
1
F
1
A), 575 (N
2
F
1
),
778 (N
3
F
1
), 2451 (H
3
N
6
F
4
A)
core-(a1–6)-fucosylation, 2 LDNF-LDNF-
antennae, 1 LDNF- antenna
a
Protonated fragment.
b

Sodium adducts of the native glycans were registered by MALDI-TOF-MS (average masses).
M. Wuhrer et al. S. mansoni N-glycans with dimeric LacdiNAc
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 353
nano-LC-MS ⁄ MS indicated the attachment of one
fucose at the subterminal HexNAc with a second
fucose linked to the fourth HexNAc of the chain
(Fig. 7). These data are in agreement with a fucosylat-
ed version of the diLDN structure, namely a diLDNF
motif {GalNAc(b1–4)[Fuc(a1–3)]GlcNAc(b1–3)Gal-
NAc(b1–4)[Fuc(a1–3)]GlcNAc(b1-} as the antenna
structure. In order to corroborate these findings, the
permethylated N-glycan species H
3
N
8
F
4
A was subjec-
ted to linkage analysis, i.e. hydrolysis, reduction and
peracetylation, followed by GC-MS analysis of the
obtained partially methylated alditol acetates using
electron-impact ionization (Fig. 8). In addition to
2-substituted mannose, 3,6-disubstituted mannose and
4-substituted GlcNAc, which are in accordance with
the trimannosyl core structure, linkage analysis revealed
terminal Fuc as well as the following N-acetylhexosa-
mine variants: terminal GalNAc, 3-substituted Gal-
NAc and 3,4-disubstituted GlcNAc (Fig. 8). Notably,
terminal GlcNAc was not detected. In conclusion, link-
age analysis data were in line with the postulated

LDNF and diLDNF antennae (Figs 7 and 9).
Furthermore, in order to obtain detailed information
about the attachment of diLDN to upper and ⁄ or lower
branch antennae for the above-mentioned isomer 3
(Figs 4 and 5C), fraction 26 was subjected to a-man-
nosidase treatment, and partial removal of 1 hexose
from the H
3
N
6
F
1
A isomer 1 was indicated by
MALDI-TOF-MS (not shown). In order to determine
the substitution position of the b-linked core mannose
in isomer 3 after removal of the terminal mannose, the
sample was subjected to permethylation and linkage
407.1
610.3
732.4
813.4
914.2
#
996.1
#
1097.3
#
1179.6
1225.8
1300.5

1381.1
1584.6
1786.8
1990.7
2193.6
0022000200810061004100210001008006004
364.2
429.2
510.3
593.6
632.2
713.2
753.2
805.6
842.8
#
915.7
#
1017.4
#
1046.3
#
1119.4
#
1147.7
#
1211.8
1256.4
1402.5
1459.3

1524.5
1605.5
1662.5
1727.5
1808.6
1930.7
1995.0
000200810061004100210001008006004
A
A
A
835.2
A
A
A (SM/SM
z/
m
]H2+M[)8911
+2
B (SM/SM
z/
m
]aN2+M[)0221
+2
Intensity
z/m
A
A
A
Fig. 6. Fragment ion spectra of the double-protonated (A) and double-sodiated (B) N-glycan species H

3
N
8
F
1
A. Spectra where obtained by
RP-nano-LC-ESI-IT-MS ⁄ MS of the 2-aminobenzamide-labelled glycans of fraction 30. The deduced structure is boxed. Yellow square, N-ace-
tylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; double-headed arrow
with dashed line, deoxyhexose; #, double-sodiated fragment.
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
354 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
analysis (not shown). In addition to 3,6-disubstituted
mannose and 2-substituted mannose, which belong to
the conventional trimannosyl core, 3-subsituted man-
nose and 6-substituted mannose were detected in a
ratio of  1 : 2. These monosaccharide derivatives arise
from isomer 3 after removal of the terminal a-linked
mannose. This would indicate that isomer 3 is a mix-
ture of monoantennary structures carrying the diLDN
motif on the lower branch (a1–3-linked mannose;
 30%) and on the upper branch (a1–6-linked man-
nose;  70%). Furthermore, terminal GalNAc, 4-sub-
stituted GlcNAc, 3-substituted GalNAc and
3,4-disubstituted GlcNAc were registered, which is in
accordance with the postulated antennae structures of
isomer 2 and isomer 3 occurring as a mixture in frac-
tion 26 (Figs 4 and 5B,C).
Proposed structures
Whereas the two N-glycans of composition H
3

N
6
F
1
(isomer 3) and H
3
N
8
F
4
(see above) were studied in
detail revealing diLDN antennae and diLDNF anten-
nae, respectively, the structural data obtained for the
other N-glycans listed in Table 1 comprised mainly
nano-LC-MS ⁄ MS analyses. Because the whole group
of N-glycans exhibited consistent patterns of character-
250.2
442.1
636.4
701.4
946.5
1027.6
1096.3
3
-N
1172.2
1197.1
1300.7
1365.7
1400.6

1423.3
2
1455.5
2
-NFA
1503.2
1632.9
2
-N
1676.8
1900.1
1964.9
0022000200810061004100
2
100010080060
0
4002
687.4
956.4
3
-N
2
F
1010.1
1113.8
A
A
A
1090.5
2

-N
4
F
2
A
dna
z/m
A SM
2
)2811(
B
SM
3
2811( → )3241
Intensity
000200510001005
687.4
767.0
857.8
946.6
1054.5
1082.6
1116.5
2
1161.3
1197.7
1423.7
1899.1
2144.2
2210.0

636.3
1293.1
2
1479.9
1321.1
2
-H
A
A
701.4
A
A
A
1950.9
1286.4
Fig. 7. Fragment ion analysis of the triple-sodiated, permethylated N-glycan H
3
N
8
F
4
A. (A) Fragment ion spectrum obtained by RP-nano-LC-
ESI-IT-MS ⁄ MS of the permethylated 2-aminobenzamide-labelled glycan from subfraction 39–4. (B) MS
3
of the double-charged precursor at
m ⁄ z 1423. The deduced structure is boxed. Yellow square, N-acetylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose;
red triangle, fucose; A, 2-aminobenzamide.
M. Wuhrer et al. S. mansoni N-glycans with dimeric LacdiNAc
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 355
istic fragment ions revealing chains of four HexNAc

residues as antenna structures carrying up to two fu-
cose residues per antenna (Table 1), we postulate that
these glycans also have antennae based on the diLDN
motif. Structures are proposed as shown in Fig. 9
based on both the data summarized in Table 1, and
the detailed analysis of compounds H
3
N
6
F
1
(isomer 3)
and H
3
N
8
F
4
.
Discussion
We describe a very heterogeneous group of N-glycans
expressed by adult worms of the human parasite
S. mansoni, which feature repeats of LDN units. These
units can also carry a fucose in the 3-position of
N-acetylglucosamine residues, resulting in repetitive
LDNF units. Repetitive LDN and LDNF structures
have not previously been described in natural sources.
Indications that such structures might occur in C. ele-
gans were given by the heterologous expression of a
C. elegans b1–4-N-acetylgalactosaminyltransferase in

CHO Lec 8 cells leading to the production of oligo-
LDN chains and, upon coexpression of an a1–3-fuco-
syltransferase, oligo-LDNF chains [23]. Whereas
Kawar et al. [23] found up to seven LDN units and up
to five LDNF units per N-glycan chain, we found up
to five LDN(F) units on schistosome N-glycans, with a
maximum of four HexNAc residues in a row [di-
LDN(F) antenna structures]. The differences in chain
length might be due to the acceptor specificities of the
involved b1–3-N-acetylglucosaminyltransferases, which
have yet to be characterized in the case of schisto-
somes as well as the CHO cells used for the expression
of the C. elegans GalNAc T [23]. The occurrence
of LDN(F) repeats parallels the poly(LacNAc) and
poly(Lewis X) chains found on N-glycans from total
S. mansoni worm glycoproteins [2–5,24], O-glycans of
the circulating cathodic antigen of S. mansoni worms
[2–5,25] as well as on glycoproteins and glycolipids of
granulocytes [26,27]. The structural similarities of par-
tially fucosylated LDN repeats and LacNAc repeats
might be paralleled by the enzymatic repertoire recrui-
ted for these biosyntheses: both the b3-GlcNAc trans-
ferase and the a3-Fuc transferase are likely to be
shared by the two biosynthetic pathways.
Many carbohydrate epitopes from schistosomes,
such as FLDN(F) {Fuc(a1–3)GalNAc(b1–4)[±
Fuc(a1–3)]GlcNAc(b1-)}, LDN(F), and Lewis X, have
been shown to be antigenic and several also appear to
be targets for the innate immune system through
recognition by human lectins [2–5,7,8,19,28–31]. In

particular, LDN(F) units, repeats of which are des-
cribed here, have been shown to be target of the host
immune response in schistosomiasis [19,29,38,39].
Despite the pronounced structural similarity between
epitopes such as Lewis X and LDNF, the N-acetyl
substitution at the 2-position of the Gal residue being
the only difference, antibodies to Lewis X or LDNF
5404
)n
im(e
mi
T
detutitsbusid-4,3
cANclG
l
anim
r
et
cANlaG
detutitsb
us-4
c
ANclG
de
tutits
bus-3
cANlaG
lanim
r
et

cANclG
*
*
*
*
A
Fig. 8. Linkage analysis of the N-glycan H
3
N
8
F
4
A. The extracted-ion chromatogram of m ⁄ z 158 indicates the HexNAc species, which were
identified based on retention times and electron-impact fragmentation patterns. The elution position of terminal GlcNAc as determined with
an authentic standard is indicated by an arrow. The 2AB-labelled, innermost GlcNAc is not detected in linkage analysis. Yellow square, N-ace-
tylgalactosamine; blue square, N-acetylglucosamine; green circle, mannose; red triangle, fucose; A, 2-aminobenzamide; *, contaminant.
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
356 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
exhibit exclusive specificity. Using surface plasmon res-
onance, it was shown that an Lewis X monoclonal
antibody selectively bound to a Lewis X neoglycopro-
tein, but not to LDNF, and vice versa (Fig. 2 in Van
Remoortere et al. [36]). Moreover, in a chimpanzee
S. mansoni-infection model, antibodies against Lewis X
showed a time course which was markedly different
from those observed for anti-LDN and anti-LDNF
sera [19]. Finally, a recent study in schistosome-infec-
ted mice has indicated an intense humoral immune
response to dimeric and trimeric Lewis X compared
with monomeric Lewis X [37]. Differential recognition

(cANla
G β (cANclG)4-1 β (naM)2-1 α )3/6-1
(naM α )
6/3-1
(naM β (cANclG)4-1 β cANclG)4-1 β nsA-1
(cuF α )6-1(cANla
G
β (cAN
c
lG)4-1 β )3
-
1
(cuF± α )3
-
1
(cuF± α )3-1
H
3
N
6
F
3-1
(cANlaG β (cANclG)4-1 β (naM)2-1 α )3/6-1
(cANc
l
G β (naM)2-1 α )6/3-1
(naM β (cANc
l
G)4-1 β cANclG)4-1 β nsA-1
(cuF α )6-1(c

A
NlaG β (cANclG)4-1 β )3-1
H
3
N
7
F
1
(cA
N
laG β (cANclG)4-1 β
(
naM
)
2-
1
α )3/6-1
(la
G
β (c
A
Ncl
G
)4-1 β
(n
aM
)2-
1
α )6/3-
1

(naM β (cANclG)4
-
1 β
c
AN
c
lG)4
-
1 β nsA-1
(cuF α )6-1(cANlaG β (cANc
l
G
)4
-1 β )3-1
(cuF α
)3
-1
(
c
u
F α
)3-
1
(cuF α )3-
1
H
4
N
7
F

4
GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6)
GalNAc(β1-4)GlcNAc(β1-2)Man(α1-3)
Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn
Fuc(α1-6)
GalNAc(β1-4)GlcNAc(β1-3)
±Fuc(α1-3)
GalNAc(β1-4)GlcNAc(β1-3)
±Fuc(α1-3)
±Fuc(α1-3)
±Fuc(α1-3)
H
3
N
10
F
1-5
GalNAc(β1-4)GlcNAc(β1-2)Man(α1-6/3)
GalNAc(β1-4)GlcNAc(β1-2)Man(α1-3/6)
Man(β1-4)GlcNAc(β1-4)GlcNAcβ1-Asn
Fuc(α1-6)GalNAc(β1-4)GlcNAc(β1-3)
±Fuc(α1-3)
±Fuc(α1-3)
±Fuc(α1-3)
H
3
N
8
F
1-4

Fig. 9. Summary of deduced structures.
M. Wuhrer et al. S. mansoni N-glycans with dimeric LacdiNAc
FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS 357
of monomeric, dimeric and trimeric Lewis X was also
shown with various monoclonal antibodies [37]. In
conclusion, the rather minor structural variations
found on schistosome glycoconjugates can have a pro-
found effect on their immunological recognition. Thus,
differences in antigenicity between monomeric LDN(F)
and oligomers thereof can be foreseen, and the
LDN(F) repeats described here are expected to be im-
munogenic and involved in host–parasite interaction.
Based on the findings of Kawar et al. [23], LDN
and LDNF repeats might not be restricted to the trem-
atode S. mansoni, but are likely to be found also on
glycoproteins of C. elegans and possibly other nema-
todes. Furthermore, future experiments will answer
the question whether mammals likewise produce these
oligo-LDN(F) antenna structures. This question
should be addressed by studying both the fine specifici-
ties of potentially involved enzymes as well as by struc-
tural studies of the N-glycosylation of LDN-expressing
tissues [20,21].
Experimental procedures
(Glyco-)protein extraction
S. mansoni worms were obtained by perfusion of infected
hamsters and stored at )80 °C until use. Adult worms were
homogenized in 1 mL of water (1 vol.Æ100 mg
)1
wet weight

of worms). In order to delipidize samples, methanol and
chloroform were sequentially added (5 vol. each). The
supernatant was removed after centrifugation, and the
extraction was repeated. In order to extract (glyco-)pro-
teins, the pellet was suspended in phosphate-buffered saline
(35 mm sodium phosphate, pH 7.6, 0.85% NaCl). SDS and
2-mercaptoethanol were added to final concentrations of
1% (w ⁄ v) and 0.5%, respectively. The samples were incuba-
ted for 10 min at 100 °C, allowed to cool to room tempera-
ture, and Chaps (Fluka/Sigma-Aldrich, Zwijndrecht, The
Netherlands) was added to a final concentration of 1%
(w ⁄ v). Samples were centrifuged, and supernatants were
subjected to PNGase F treatment. These experiments were
carried out in accordance with EC Council Directive
(89/609/EEC) and after approval of the animal experiment
commitee (DEC) of the Leiden University Medical Center.
Glycan release
S. mansoni (glyco-)proteins were incubated with PNGase F
(2 mUÆ100 mg
)1
wet weight; Roche Diagnostics, Mannheim,
Germany) overnight at 37 °C. For the purification of the
released glycans, samples were first applied to a reverse-phase
cartridge (500 mg of Bakerbond octadecyl; Baker, Phillips-
burg, NJ). Flow-through and wash (5 mL of water) were
then applied to a carbon cartridge (150 mg Carbograph;
Alltech, Deerfield, IL). After washing with water (5 mL),
glycans were eluted with 25% aqueous acetonitrile (5 mL).
Released glycans were detected by MALDI-TOF-MS.
MALDI-TOF-MS

Glycan samples were analysed by MALDI-TOF-MS using
an Ultraflex mass spectrometer (Bruker Daltonics, Bremen,
Germany) in the positive reflectron mode with 6-aza-2-thio-
thymine (5 mgÆmL
)1
; Sigma, St. Louis, MO) as matrix.
Labelling and fractionation
Glycans released with PNGase F were tagged with 2AB by
reductive amination as outlined previously [40]. The reac-
tion mixture was applied to a carbon cartridge (Alltech,
Deerfield, IL), and the 2AB-labelled glycans were eluted
with 5 mL of 25% acetonitrile. The acetonitrile content was
reduced under a stream of nitrogen, and the samples were
lyophilized.
Fractionation by normal-phase HPLC
2AB-labelled glycans were fractionated by normal-phase
HPLC on a TSK-Amide 80 column (4 · 250 mm; Tos-
ohaas, Montgomeryville, PA) at 0.4 mLÆmin
)1
. Solvent A
was 5 mm formic acid adjusted to pH 4.4 with ammonia,
which is a modification of a previously published separation
system [41]. Solvent B was 20% of solvent A in acetonitrile.
The following gradient conditions were used: at time t ¼
0 min, 100% solvent B; t ¼ 152 min, 52.5% solvent B; t ¼
155 min, 0% solvent B; t ¼ 162 min, 0% solvent B; and
t ¼ 163 min, 100% solvent B. The total run time was
180 min. Samples were injected in 80% acetonitrile.
Because of the large amounts of material injected, fluores-
cence was detected at 280 nm ⁄ 500 nm instead of the rou-

tinely used 360 nm ⁄ 425 nm in order to avoid saturation of
the detector. Fractions were collected manually and ana-
lysed by MALDI-TOF-MS.
Fractionation by RP-HPLC
2AB-labelled glycans were fractionated by RP-HPLC on a
Hypersil ODS 3 lm(2· 250 mm; Thermo Electron Corp.,
Waltham, MA) at 0.2 mLÆmin
)1
. Solvent A was 0.4%
acetonitrile, 0.1% formic acid. Solvent B was 95% acetonit-
rile, 0.1% formic acid. The following gradient conditions
were used: at time t ¼ 0 min, 5% solvent B; t ¼ 5 min,
5% solvent B; gradient to t ¼ 30 min, 50% solvent B; t ¼
31 min, 100% solvent B; and t ¼ 36 min, 100% solvent B,
t ¼ 37 min, 5% solvent B. Total run time was 60 min.
Fluorescence was detected at 360 nm ⁄ 425 nm. Fractions
were collected manually and analysed by MALDI-
TOF-MS.
S. mansoni N-glycans with dimeric LacdiNAc M. Wuhrer et al.
358 FEBS Journal 273 (2006) 347–361 ª 2005 The Authors Journal compilation ª 2005 FEBS
Nano-LC-MS/MS
2AB-labelled glycans were separated on a PepMap col-
umn (75 lm · 150 mm; Dionex ⁄ LC Packings, Amster-
dam, the Netherlands) using an Ultimate nano-LC system
(Dionex ⁄ LC Packings) equipped with a Switchos guard
column system (Pepmap guard column, 300 lm · 10 mm).
The system was equilibrated with eluent A (H
2
O ⁄ aceto-
nitrile 95 : 5, v ⁄ v, 0.1% formic acid) at a flow rate of

150 nLÆmin
)1
. After injecting the sample, a linear gradient
to 50% eluent B (H
2
O ⁄ acetonitrile 5 : 95, v ⁄ v, containing
0.1% formic acid) in 30 min was applied, followed by a
final wash with 100% B for 5 min. The system was
directly coupled to an Esquire high capacity trap (HCT)
ESI-IT-MS (Bruker) equipped with an online nano-spray
source operating in the positive-ion mode. For electro-
spray (900–1200 V), capillaries (360 lm OD, 20 lmID
with 10 l m opening) from New Objective (Cambridge,
MA) were used. The solvent was evaporated at 165 °C
with a nitrogen stream of 5 LÆmin
)1
. Ions from m ⁄ z 50
to 2000 were registered. Automatic fragment ion analysis
was enabled, resulting in MS ⁄ MS spectra of the most
abundant peaks. In order to register predominantly
sodium adducts by MS, part of the analyses were per-
formed after addition of 0.8 mm NaOH to solvent A.
For the analysis of permethylated glycans, the column
was conditioned with 15% eluent B. After injection, a
linear gradient to 70% eluent B in 30 min was applied
followed by a wash with 100% B for 5 min.
a-Mannosidase treatment
2AB-labelled glycans (50 pmol to 1 nmol) were treated with
a-mannosidase from jack beans (100 mU; Sigma) in 50 lL
50 mm sodium acetate buffer, pH 5.0 for 18 h at 37 °C.

The reaction mixture was applied to a carbon cartridge
(Alltech), and the 2AB-labelled glycans were eluted with
5 mL of 25% acetonitrile, analysed by MALDI-TOF-MS,
and subjected to nano-LC-MS ⁄ MS.
Permethylation and linkage analysis
2AB-labelled glycans were permethylated [42] and analysed
by nano-LC-MS ⁄ MS. For linkage analysis, permethylated
glycans were hydrolyzed (4 m trifluoroacetic acid, 4 h,
100 °C), and partially methylated alditol acetates obtained
after sodium borohydride reduction and peracetylation
were analysed by capillary GLC-MS using electron-impact
ionization [43].
Acknowledgements
We thank Ria van den Heuvel for performing GC-MS
analyses.
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