Tài liệu Báo cáo khóa học: Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core doc

Determination by electrospray mass spectrometry and
1
H-NMR
spectroscopy of primary structures of variously fucosylated neutral
oligosaccharides based on the
iso
-lacto-
N
-octaose core
Heide Kogelberg
1
, Vladimir E. Piskarev
2
, Yibing Zhang
1
, Alexander M. Lawson
1
and Wengang Chai
1
1
MRC Glycosciences Laboratory, Imperial College Faculty of Medicine, Northwick Park Institute for Medical Research, Harrow,
Middlesex, UK;
2
Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia
We have isolated a nonfucosylated and three variously
fucosylated neutral oligosaccharides from human milk that
are based on the iso-lacto-N-octaose core. Their structures
were characterized by the combined use of electrospray
mass spectrometry (ES-MS) and NMR spectroscopy. The
branching pattern and blood group-related Lewis deter-
minants, together with partial sequences and linkages of

these oligosaccharides, were initially elucidated by high-
sensitivity ES-MS/MS analysis, and then their full structure
assignment was completed by methylation analysis and
1
H-NMR. Three new structures were identified. The
nonfucosylated iso-lacto-N-octaose, Galb1–3GlcNAcb1–
3Galb1–4GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc,
has not previously been reported as an individual oligo-
saccharide. The monofucosylated and trifucosylated
iso-lacto-N-octaose, Galb1–3GlcNAcb1–3Galb1–4(Fuca1–3)
GlcNAcb1–6[Galb1–3GlcNAcb1–3]Galb1–4Glc and Galb1–
3(Fuca1–4)GlcNAcb1–3Galb1–4(Fuca1–3)GlcNAcb1–
6[Galb1–3(Fuca1–4)GlcNAcb1–3]Galb1–4Glc, both con-
taining an internal Le
x
epitope, are also novel structures.
Keywords: electrospray mass spectrometry; human milk;
oligosaccharide; NMR.
A role for carbohydrates in cellular events has long been
hypothesized, although strong evidence for this has only
emerged over the last two decades. Awareness of the
biological function of oligosaccharide chains in glycopro-
teins, glycolipids and proteoglycans has intensified as an
increasing number of examples have been reported that
reveal that carbohydrate structures participate in various
biological events in addition to modifying protein function.
One of the early demonstrations of the role of carbo-
hydrates in recognition was binding of the influenza virus to
red blood cells via sialic acid [1], and later by work on the
chemical basis of the antigenicity of polysaccharides and

of the well-known ABO (H) blood-group system [2,3],
in which specificity is determined by oligosaccharide
sequences. Carbohydrates are well placed to act in cellular
recognition as many cells are surrounded by an oligosac-
charide layer made from cell-associated glycoconjugates,
which often overshadows protein and lipid components on
the cell surface. Specific oligosaccharide sequences, such
as the type 1 (Galb1–3GlcNAc)/type 2 (Galb1–4GlcNAc)
chains and the blood group-related antigens bearing the
H(Fuca1–2Galb1–3/4GlcNAc), Lewis
a
[Le
a
,Galb1–
3(Fuca1–4)GlcNAc] and Lewis
x
[Le
x
,Galb1–4(Fuca1–
3)GlcNAc] determinants, occur naturally as structural
elements of free oligosaccharides or on the carbohydrate
chains of glycoproteins and glycolipids and comprise
recognition motifs for cell–cell and cell–matrix interactions
[4,5].
Human milk is a unique source of diverse oligosaccha-
rides, and more than 80 have been isolated and sequences
assigned [6]. Many of these structures are closely related to
the carbohydrate chains of glycoproteins and glycolipids [7].
These diverse oligosaccharide sequences may also serve as
cell differentiation and tumour antigens [5]. Milk oligosac-

charides are considered to play a part in the inhibition of
bacterial adhesion to epithelial surfaces, as they are able to
mimic the binding epitope of the epithelial receptor [8].
Also, milk contains oligosaccharides that resemble structures
recognized by the cell–cell adhesion molecules, the selectins,
suggesting a role in inflammatory processes [8,9]. Human
milk has also been used as a rich source of oligosaccharides
to map the fine binding specificity of E-selectin [10].
In contrast with oligonucleotides and peptides, oligosac-
charides can be branched, and hence a relatively simple set
of monosaccharides can form a huge number of complex
structures. A greater degree of structural complexity
produced by branching is the norm for naturally occurring
carbohydrates, and often a branched sequence carrying two
or more recognition motifs is more potent [11,12]. Free
oligosaccharides from human (milk, urine and infant faeces)
have a common lactose (Galb1–4Glc) core. It can be
extended, for example, at the 4-position of the Gal as a
linear sequence or at its 3,6-positions as a branched
sequence. The linear and branched chains are often
Correspondence to W. Chai, MRC Glycosciences Laboratory,
Imperial College Faculty of Medicine, Northwick Park Institute for
Medical Research, Watford Road, Harrow, Middlesex HA1 3UJ,
UK. Fax: + 44 20 8869 3253, Tel.: + 44 20 8869 3252,
E-mail:
Abbreviations: CID, collision-induced dissociation; ES-MS, electro-
spray mass spectrometry; iLNO, iso-lacto-N-octaose; Le
a
,Lewisa;
Le

x
, Lewis x; PMAA, partially methylated alditol acetate; rOe,
rotating frame nuclear Overhauser enhancement.
(Received 4 December 2003, accepted 3 February 2004)
Eur. J. Biochem. 271, 1172–1186 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04021.x
fucosylated to varying degrees to form several of the blood
group-related antigens.
Methods for detailed characterization of these recogni-
tion motifs are important in modern structural cell biology
to derive structure/function relationships, particularly in the
postgenome era, in order to understand post-translational
glycosylation and its function. Expansion of our knowledge
on the repertoire of carbohydrate structures, the ÔglycomeÕ,
and investigation of oligosaccharide epitopes involved
in carbohydrate–protein interactions require their detailed
isolation and structural determination. With small amounts
of material (e.g. a few picomoles), no single analytical
technique is capable of the complete characterization of an
oligosaccharide structure. Structure elucidation is therefore
usually achieved by using several different techniques, of
which MS and NMR are two of the most powerful.
Previously, we demonstrated the distinction of chain type
and blood-group type (such as Le
a/x
and Le
b/y
) of underi-
vatized oligosaccharides by negative-ion electrospray mass
spectrometry (ES-MS) with collision-induced dissociation
(CID) and MS/MS scanning with low picomole sensitivity

[13,14]. Several characteristic fragmentations are useful
for obtaining detailed structural information. The double
glycosidic D-type cleavage [13,14] is unique to 3-linked
GlcNAc and Glc residues, whereas
0,2
A-type cleavages [15]
only occur with 4-linked GlcNAc and Glc, resulting in
fragment ions, indicating type 1 and type 2 chains or blood
group types. For a 3-linked GlcNAc or Glc, the D-type
cleavage occurs at the glycosidic bonds of both reducing and
nonreducing sides of the residue, by combined C-type and
Z-type cleavages (see Results for discussion and Figs 1–4 for
illustration). For -3GlcNAc- without further substitution, a
fragment at m/z 202 is obtained. If a Fuc is present at the
4-position of the -3GlcNAc- (e.g. in the case of an Le
a
determinant), a fragment at m/z 348 (202 +146) results,
whereas if the 4-position is substituted by Gal (e.g. in the
case of an Le
x
determinant), a unique fragment at m/z 364 is
produced. Therefore, a D-fragment at m/z 202 indicates a
Fig. 1. Product-ion spectra of iLNO with [M-H]
–
(A) and [M-2H]
2–
(B) as precursors. The structure of iLNO is shown to indicate the proposed
fragmentation. The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with ÔhÕ are fragments
produced by dehydration of the major ions.
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1173

type 1 chain, whereas an
0,2
A-ion doublet at m/z 281/263
indicates a type 2 chain. D-ions at m/z 348 or m/z 364 are
characteristic of either terminal Le
a
or Le
x
determinants,
respectively [13]. We also established a method for core-
branching pattern analysis using CID MS/MS of singly and
doubly charged molecular ions [14]. These spectra give
complementary structural information. In the CID spectra
of [M-H]
–
, fragment ions from the 6-linked branch are
dominant, and those from the 3-linked branch are absent,
whereas fragment ions from both branches occur in the
product-ion spectra of [M-2H]
2–
. This allows us to distin-
guish between fragment ions derived from either the 3- or
the 6-branch and to deduce the branching pattern and also
assign structural details of the 3- and 6-branches.
Although MS is the more sensitive method of structural
analysis, NMR spectroscopy is the choice for more
complete assignment of carbohydrate structure when suffi-
cient material is available. In this report, we demonstrate
our strategy of the combined use of ES-MS and NMR for
analysis of the core-branching pattern and full sequence

assignment of four oligosaccharides isolated from human
milk. These are variously fucosylated structures based
on the iso-lacto-N-octaose core, of which three are novel
sequences.
Materials and methods
Isolation and purification of oligosaccharides
Oligosaccharides were isolated from human milk obtained
from a healthy 25-year-old woman, blood group B
secretor, Le
b
positive, giving negative reaction in hepatitis
B and HIV tests. Written consent was obtained from this
volunteer for analysis of the milk sample. Fat was
removed by centrifugation at 4 °C (5000 g,30min)and
proteins by precipitation with cold 50% (v/v) acetone.
Oligosaccharides were separated from lactose on a
Sephadex G-25 column (5 · 90 cm), and then neutral
Fig. 2. Product-ion spectra of MFiLNO with [M-H]
–
(A) and [M-2H]
2–
(B) as precursors. The structure of MFiLNO is shown to indicate the
proposed fragmentation. The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with ÔhÕ are
fragments produced by dehydration of the major ions.
1174 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
from acidic oligosaccharides on a Dowex 1 (· 2; 100–200
mesh; acetate form) column. Further gel-filtration chro-
matography was carried out on a Fractogel HW-40(S)
column (5 · 90 cm). Oligosaccharides were eluted from
the gel-filtration and ion-exchange columns with distilled

water. The partially resolved octasaccharide to undeca-
saccharide fractions were further fractionated by normal-
phase HPLC on a preparative Separon amino column
(10 · 250 mm) by elution with 50% (v/v) acetonitrile to
give octasaccharide (F8), nonasaccharide (F9), decasac-
charide (F10) and undecasaccharide (F11) fractions. Each
subfraction was purified by reverse-phase HPLC on a
Zorbax octadecyl column (10 · 250 mm) by elution with
water. The octasaccharide iso-lacto-N-octaose (iLNO) was
obtained from F8, monofucosyl iLNO (MFiLNO) from
F9, difucosyl iLNO (DFiLNO) from F10, and trifucosyl
iLNO (TFiLNO) from F11. Repeated normal-phase
HPLC was carried out to ensure the purity of each
oligosaccharide fraction before their analyses by ES-MS
and
1
H-NMR spectroscopy.
Methylation analysis
After initial reduction with NaBD
4
, oligosaccharides were
methylated, hydrolysed, reduced, and acetylated as des-
cribed previously [16]. GC-MS analysis of the partially
methylated alditol acetates was performed on a Thermo-
Quest Trace system using a 15-m RTX-5 capillary column.
The initial column temperature was 50 °C programmed to
100 °Cat25°CÆmin
)1
,to220°Cat5°CÆmin
)1

and to
310 °Cat10°CÆmin
)1
.
ES-MS
Negative-ion ES-MS and CID MS/MS were carried out on
a Micromass (Manchester, UK) Q-Tof mass spectrometer.
Nitrogen was used as desolvation and nebuliser gas at a flow
rate of 250 LÆh
)1
and 15 LÆh
)1
, respectively. Source tem-
perature was 80 °C, and the desolvation temperature
150 °C. Typically, a cone voltage of 80 V was used for
CID MS/MS of singly charged ions [M-H]
–
and 50 V for
Fig. 3. Product-ion spectra of DFiLNO with [M-H]
–
(A) and [M-2H]
2–
(B) as precursors. The structure of DFiLNO is shown to indicate the
proposed fragmentation. The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with ÔhÕ are
fragments produced by dehydration of the major ions.
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1175
doubly charged ions [M-2H]
2–
. The capillary voltage was
maintained at 3 kV. Product-ion spectra were obtained

from CID using argon as the collision gas at a pressure of
0.17 MPa. The collision energy was adjusted to 23–43 V for
optimal fragmentation and, typically, 40–43 V was used for
CID of [M-H]
–
, and 23–27 V for [M-H]
2–
.Ascanrateof
1.5sperscanwasusedforbothES-MSandCIDMS/MS
experiments, and the acquired spectra were summed for
presentation.
For analysis, oligosaccharides were dissolved in acetonit-
rile/water (1 : 1, v/v), typically at a concentration of
5–10 pmolÆlL
)1
,ofwhich5lL was loop-injected. Solvent
(acetonitrile/1 m
M
ammonium bicarbonate, 1 : 1, v/v) was
delivered by a Harvard syringe pump (Harvard Apparatus,
Holliston, MA, USA) at a flow rate of 10 lLÆmin
)1
.
Alternatively, 0.5–1 lL sample solution was placed in a
capillary needle for the nanospray experiment.
NMR spectroscopy
Oligosaccharides were coevaporated with
2
H
2

O (99.9
atom%
2
H
2
) twice by lyophilization and dissolved in
500 lL high-quality
2
H
2
O (100.0 atom%
2
H
2
), containing
0.1 lL acetone.
NMRspectrawereacquiredonVarian(PaloAlto,CA,
USA) Unity plus-500 (500.07 MHz
1
H), Unity-600
(599.89 MHz
1
H) and Unity-800 (800.27 MHz
1
H) spec-
trometers and processed with standard Varian software.
The observed
1
H chemical shifts were reported relative to
internal acetone (2.225 p.p.m.). The NMR spectra were

recorded at 15 °CforiLNO,10°CforMFiLNO,13°Cfor
DFiLNO, and 27 °C for TFiLNO. The temperatures were
chosen in order to place the H
2
O signals with minimal
disturbance to carbohydrate protons. For MFiLNO, for
example, a temperature of 10 °CplacedtheH
2
Osignal
optimally downfield from the Fuc IX H5 proton. For
TFiLNO, the same temperature would have resulted in total
overlap of the H
2
O signal with the Fuc X and XI H5
protons. Therefore the temperature of 27 °C was chosen for
TFiLNO. This placed the H
2
O signal between Fuc IX H5
and the GlcNAc V and VII H1 protons; nevertheless, the
Fuc IX H5 was slightly obscured.
2D phase-sensitive TOCSY spectra were recorded at
mixing times of 10, 30, 50, 70 and 140 ms. A spectral width
of 3500 Hz was used in both dimensions, with eight scans
per increment. 2D phase-sensitive ROESY experiments
were performed with a mixing time of 300 ms, a spectral
width of 8000 Hz in both dimensions, and 16 scans per
increment. The spectrum offset was set 1.5 p.p.m. to
lower field of the most downfield shifted proton, Glc1a,
to minimize TOCSY transfer. The raw data sets of the
homonuclear 2D experiments typically consisted of

4K · 256 complex data points.
Results
Determination of branching pattern and blood group
determinants by ES-MS/MS
Negative-ion CID ES-MS/MS product ion scanning with
the strategy established previously [13,14] was used first for
analysis of the branching patterns and blood group
determinants with low (picomole) amounts of the underi-
vatized oligosaccharides. The significant aspects of this
strategy are the combined use of CID MS/MS of singly and
doubly charged molecular ions as precursors to deduce the
Fig. 4. Product-ion spectrum of D
2b-5
ion m/z 1183 of DFiLNO as precursor (A) and the D
2b-5
ion m/z 1037 of a contaminant in DFiLNO as the
precursor (B).
1176 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
branching pattern, sequence, and partial linkages, and
assign structural details of each branch (e.g. the 3- and
6-branches). Blood group H, Le
a
/Le
x
and Le
b
/Le
y
deter-
minants, together with type 1 and type 2 chains, can be

determined from their unique fragment ions.
iLNO. The singly charged molecular ion [M-H]
–
at m/z
1436 (Fig. 1A) and doubly charged [M-2H]
2–
at m/z 717.8
(Fig. 1B) are consistent with an octasaccharide of compo-
sition Hex
5
HexNAc
3
. The approximate relative proportions
of partially methylated alditol acetates (PMAAs) from
methylation analysis (Table 1) are in agreement with this
and indicate that the monosaccharide residues include one
reducing terminal 4-linked Glc, four Gal (two terminal, one
internal 3-substituted and one 3,6-disubstituted) and three
GlcNAc (one 4-linked and two 3-linked). As established
previously, the product-ion spectrum of [M-H]
–
only shows
the fragment ions on the 6-linked branch. The C-type ions
C
1a
,C
2a
,C
3a
,C

4a
and C
5
(see the fragmentation scheme in
Fig. 1) are in agreement with a 6-linked branch Gal-
GlcNAc-Gal-GlcNAc-, while ions D
2b-5
and
0,3
A
5
at m/z
891 and 819, respectively, define a 3,6-branched core Gal
[14]. The mass difference between the ion C
5
(m/z 1274) and
[M-H]
–
(m/z 1436) indicates a Hex at the reducing terminus.
The characteristic
0,2
A
6
doublet (m/z 1376/1358) and
2,4
A
6
(m/z 1316) ions confirm this to be a -4Glc [13,14]. Thus, a
reducing terminal core 3,6-disubstituted -Gal-4Glc can be
proposed, in agreement with methylation analysis. Further-

more, 3-substituted GlcNAc next to the nonreducing
terminal Gal can be deduced from a D
1a-2a
ion [13] at m/z
202 while the HexNAc linked to the core Gal is deduced to
be a -4GlcNAc from the unique
0,2
A fragmentation (ions at
m/z 646/628). Together with the monosaccharide linkage
analysis data (Table 1), this further defines the sequence to
be Gal1–3GlcNAc1–3Gal-4GlcNAc-6(Gal-GlcNAc-3)Gal-
4Glc. From the knowledge that the product ion spectrum of
[M-2H]
2–
shows fragment ions from both branches, and as
no additional fragment ions are apparent, it can be deduced
that the disaccharide sequence on the 3-branch shares the
same terminal sequence Gal1–3GlcNAc Taken together,
these features indicate iLNO to be:
MFiLNO. The singly charged molecular ion [M-H]
–
at
m/z 1582 (Fig. 2A) and doubly charged [M-2H]
2–
at m/z
790.8 (Fig. 2B) are consistent with a nonasaccharide of
composition dHex
1
Hex
5

HexNAc
3
. The approximate rel-
ative proportions of PMAAs from methylation analysis
(Table 1) are in agreement with this and indicate that the
monosaccharide residues include Fuc, in addition to one
reducing terminal 4-linked Glc, four Gal (two terminal,
one internal 3-linked and one 3,6-disubstituted) and three
GlcNAc (one 3,4-disubstituted and two 3-linked). The
location of the Fuc residue can be deduced from the CID
MS/MS fragmentation. In comparison with the spectrum
of iLNO (Fig. 1A), the D
2b-5
ion has shifted 146 Da to
m/z 1037 corresponding to the presence of a fucose on the
6-branch. The fragment ions at m/z 161, 382, 544 and 202
as B
1a
,C
2a,
C
3a
and D
1a-2a
, respectively, are the same as
those in the spectrum of iLNO (Fig. 1A). However, C
4a
has shifted from m/z 747 for iLNO to m/z 893, indicating
the Fuc is at the internal 3,4-disubstituted GlcNAc. The
absence of a

0,2
A
4a
doublet and the presence of a D
4f-4a
ion at m/z 729 (see the fragmentation scheme in Fig. 2)
are in agreement with a Fuc at the 3-position of a
GlcNAc, indicating a Le
x
determinant. Again, no addi-
tional fragment ions were revealed in the product ion
spectrum of the doubly charged precursor (Fig. 2B),
confirming that the 3-linked disaccharide branch is the
Table 1. Linkage and monosaccharide composition assignment from methylation analysis of milk oligosaccharides. PMAA, partially methylated
alditol acetate. Molar ratios are relative to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol. –, Not detected.
PMAA Linkage
Molar ratio
iLNO MFiLNO DFiLNO TFiLNO
Fucitol
1,5-di-O-acetyl Fuc1- – 0.8 1.8 3.3
Glucitol
4-mono-O-acetyl -4Glcol 1.2 1.2 1.4 0.7
Galactitol
1,5-di-O-acetyl Gal1- 2.0 2.0 2.0 2.0
1,2,5-tri-O-acetyl -2Gal1- – – – –
1,3,5-tri-O-acetyl -3Gal1- 0.4 1.2 0.9 0.9
1,2,3,5-tetra-O-acetyl -2,3Gal1- – – – –
1,3,5,6-tetra-O-acetyl -3,6Gal1- 1.0 1.2 0.9 1.7
N-Acetylglucosaminatol
1,5-di-O-acetyl GlcNAc1- – – – –

1,4,5-tri-O-acetyl -4GlcNAc1- 0.9 – – –
1,3,5-tri-O-acetyl -3GlcNAc1- 1.4 1.8 1.4 –
1,3,4,5-tetra-O-acetyl -3,4-GlcNAc1- – 0.7 2.1 3.1
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1177
same as the terminal disaccharide sequence of the
6-linked branch. Hence, this monofucosylated octasaccha-
ride, MFiLNO, can be assigned as:
DFiLNO. The singly charged molecular ion at m/z 1729
and the doubly charged ion at m/z 863.8 in the spectrum
of DFiLNO (Fig. 3) are consistent with a difucosylated
octasaccharide (dHex
2
Hex
5
HexNAc
3
) in agreement with
methylation analysis (Table 1), which also shows a mono-
saccharide composition corresponding to MFiLNO with an
additional Fuc. It is apparent that both fucose residues are
located in the 6-linked branch, as evidenced by the unique
and intense D
2b-5
ion at m/z 1183 (see the fragmentation
scheme in Fig. 3), from cleavage of the core residue Gal
(Fig. 3A), and by
0,3
A
5
at m/z 1111. The C

2a
ion at m/z 528
shows that one Fuc is at the subterminal GlcNAc, and the
characteristic D
1a-2a
ion [13] at m/z 348 is consistent with a
Fuc 4-linked to the GlcNAc of a terminal Le
a
determinant.
As the C
3a
ion is at m/z 690, this excludes the possibility of
the other Fuc being at the Gal next to the subterminal
GlcNAc. The position of the second Fuc 3-linked to the
internal GlcNAc forming an internal Le
x
determinant is
deduced from the characteristic double cleavage D
4f-4a
ion
at m/z 875. The ions at m/z 1037, m/z 729 and m/z 544 are
similar to those in the spectrum of MFiLNO (Fig. 2), in
which only one Fuc is in the 6-branch, and believed to be
from a contaminant (see below), having the same molecular
mass with one Fuc in each of the 3- and 6-branches.
Comparison of the product-ion spectra of the singly and
doubly charged molecular ion precursor (Fig. 3A,B)
show the major additional ions in the doubly charged ion
spectrum to be m/z 202 and m/z 382. The former derives
from a D

1b-2b
cleavage consistent with a 3-linked GlcNAc,
and the latter from a glycosidic C
2b
cleavage, which
supports assignment of the unsubstituted disaccharide
sequence on the 3-branch. The ion at m/z 803 is the doubly
charged
2,4
A
6
fragment, which appears as a singly charged
ion (m/z 1608) in the product-ion spectrum of the singly
charged precursor (Fig. 3A). The product-ion spectra of the
doubly charged precursors generally give more intense
fragment ion peaks.
The ions at m/z 1037, m/z 729 and m/z 544 appearing in
both spectra are similar to those in the spectra of MFiLNO
(Fig. 2), in which only one Fuc is in the 6-branch, and, as
indicated above, are believed to be from a contaminant with
the same molecular mass with one Fuc in each of the 3- and
6-branches. This is confirmed by MS/MS scanning of the
D
2b-5
ions m/z 1183 (Fig. 4A) and m/z 1037 (Fig. 4B) as
precursors, produced by cone voltage fragmentation [14].
The fragment ions of m/z 729 and m/z 544 are both from the
monofucosylated D
2b-5
ion m/z 1037 and not from the

difucosylated D
2b-5
ion m/z 1183.
Thus the major component, the difucosylated branched
octasaccharide, can be assigned as:
and the minor component as:
TFiLNO. The singly charged molecular ion [M-H]
–
at m/z
1874 (Fig. 5A) and doubly charged [M-2H]
2–
at m/z 937.0
(Fig. 5B) are consistent with a trifucosylated octasaccharide
with composition Fuc
3
Hex
5
HexNAc
3
. The approximate
relative proportions of PMAAs from methylation analysis
(Table 1) are in agreement with this and indicate that the
monosaccharide residues include three terminal Fuc1-, two
terminal Gal1-, one reducing terminal -4Glc, together with
internal monosubstituted and disubstituted residues: one
-3Gal1-, one -3,6Gal1-, three -3,4GlcNAc1 The branching
pattern and the locations of the three fucose residues can
be deduced by similar reasoning to that for the other
fucosylated analogues. The product-ion spectrum of the
singly charged precursor (Fig. 5A) is very similar to that of

DFiLNO (Fig. 3A), indicating a similarly difucosylated
tetrasaccharide sequence on the 6-branch. The remaining
Fuc is on the 3-branch, as indicated by C
5
at m/z 1712 and
D
2b-5
at m/z 1183 (Fig. 5). It can be deduced that the Fuc is
4-linked to the -3GlcNAc-, forming a second terminal Le
a
determinant, as no additional fragment ions are apparent
in the spectrum of doubly charged precursor. Hence the
trifucosylated octasaccharide contains two terminal Le
a
and
one internal Le
x
determinants with the sequence:
Completion of sequence assignment by
1
H NMR
Homonuclear NMR was carried out to verify the MS
assignment of the iLNO core structures and its variously
fucosylated analogues, and to determine their anomeric
configurations and linkages. The proton chemical shifts of
1178 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
individual monosaccharide residues were assigned with the
aid of TOCSY spectra with increasing mixing times (data
not shown). Oligosaccharide sequences were established
from interresidue rotating frame nuclear Overhauser

enhancements (rOes) in combination with chemical shifts.
iLNO. The monosaccharide composition of iLNO was
shown by methylation analysis to comprise one Glc
(reducing end), four Gal (two terminal) and three GlcNAc
residues (see above). Their
1
H chemical-shift assignments,
obtained from TOCSY spectra (Fig. 6), are given in
Table 2. Anomeric proton chemical shifts of four Gal
residues are present between 4.47 and 4.427 p.p.m., while
those of GlcNAc are between 4.71 and 4.634 p.p.m. The
reducing terminal Glc I residue (see structure in Fig. 6 and
below) is indicated by the respective chemical shifts of
the a-anomers and b-anomers at 5.22 and 4.665 p.p.m.
(Table 2). All residues are in b-anomeric linkages, as
deduced from H
1
,H
2
coupling constants between 8.0 and
8.3 Hz (Table 2).
Sequence assignment of iLNO was derived from
interresidue rOes (Table 2) and confirmed the MS assign-
ment. The reducing Glc I is deduced to be substituted at
position 4, as the b-anomer of Gal II H1 gives an rOe to
this proton. Gal II is a branching point and substituted at
positions 3 and 6, as GlcNAc III H1 gives an rOe to H6a
and H6b of Gal II, and GlcNAc VII H1 gives an rOe to
the H3 of this residue. The 3-branch is terminated by Gal
VIII which is linked to the 3-position of GlcNAc VII, as

Gal VIII H1 gives an rOe to GlcNAcVII H3. In addition,
the GalVIIIb1–3GlcNAcVII linkage is supported by
an rOe between GalVIII H1 and NAc protons of
GlcNAc VII.
Extension of the 6-branch is by a GalIVb1–4GlcNAcIII
linkage, as GalIV H1 gives an rOe to H4 of GlcNAc III. Gal
IV is further extended by a GlcNAcVb1–3GalIV linkage, as
GlcNAc V H1 gives an rOe to Gal IV H3. Finally, a
terminal Gal VI is linked b1–3 to GlcNAc V, as Gal VI H1
gives an rOe to GlcNAc V H3, and Gal VI H1 gives an rOe
to the NAc protons of GlcNAc V.
Fig. 5. Product-ion spectra of TFiLNO with [M-H]
–
(A) and [M-2H]
2–
(B) as precursors. The structure of TFiLNO is shown to indicate the proposed
fragmentation. The nomenclature used to define the cleavage is based on that introduced previously [15], and ions marked with ÔhÕ are fragments
produced by dehydration of the major ions.
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1179
The structure of iLNO is further supported from
comparisons of chemical shifts with those reported previ-
ously for similar oligosaccharides. The proton chemical
shifts of the Gal II and Glc I residues and residues on the
3-branch (VII and VIII) are almost identical with those
reported previously for an iso-lacto-N-octaose derivative
that is difucosylated on the 6-branch [17]. Furthermore the
proton chemical shifts of the structural reporter protons of
GlcNAc III are almost identical with those reported for the
GlcNAcb1–6 residue of lacto-N-hexaose [18].
Fig. 6. 1D and 2D

1
H NMR spectra (800 MHz) of iLNO, region 5.5–3.0 p.p.m., at 15 °C. Upper trace,
1
H NMR spectrum; top-left half, 300-ms
ROESY spectrum and bottom-right half, 140-ms TOCSY spectrum. The structure is shown at the top, depicting the residue labelling.
1180 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Taken together these data allow assignment of the iLNO
structure as follows:
TFiLNO. From methylation analysis, it was deduced that
TFiLNO comprises four Gal residues, three GlcNAc
residues, one reducing Glc residue, and three fucose residues
(see above).
1
H chemical-shift assignments of these residues
(Table 3) were made from TOCSY spectra (see Fig. 8) with
increasing mixing times (data not shown). The Gal and
GlcNAc residues are all in b-anomeric linkages as deduced
from H
1
,H
2
coupling constants between 7.8 and 8.0 Hz,
while the fucose residues are a-linked to their neighbouring
residues apparent from H
1
,H
2
coupling constants of 3.7 and
3.8 Hz (Table 3).
The sequence of the oligosaccharide was derived from

interresidue rOes (Table 3, Figs 7 and 8) which confirmed
the MS assignment. The reducing Glc residue is substituted
at position 4; rOes are observed between Gal II H1 and
Glc I H4 in the b-anomer. Gal II is a branching point and
substituted at positions 3 and 6, as GlcNAc III H1 gives an
rOe to H6b of this residue, while GlcNAc VII H1 gives an
rOe to H3 of Gal II. The 3-branch is further extended by a
terminal Gal VIII residue, as Gal VIII H1 gives an rOe to
GlcNAc VII H3. The Gal VIIIb1–3GlcNAc VII linkage is
further supported by the rOe between Gal VIII H1 and the
NAc protons of GlcNAc VII. GlcNAc VII is also substi-
tuted by Fuc XI, which is linked to the 4-position of this
residue, as Fuc XI H1 gives an rOe to GlcNAc VII H4. The
terminal trisaccharide, Gal VIIIb1–3(Fuc XIa1–4)GlcNAc
VII, the Le
a
epitope, also shows remote interresidue rOes of
FucXIH5andFucXICH3toGalVIIIH2.Thisisa
characteristic rOe pattern for this epitope, resulting from
stacking interactions of the Gal VIII and Fuc XI residues
[19,20].
The GlcNAc III on the 6-branch is substituted at
positions 3 and 4, as Gal IV H1 gives an rOe to H4 of
GlcNAc III, and Fuc IX H1 gives an rOe to H3 and to the
NAc protons of this residue. The trisaccharide, Gal IVb1–
4(Fuc IXa1–3)GlcNAc III, the Le
x
epitope, is further
characterized by remote rOes between Fuc IX H5 and Gal
IV H2 and between Fuc IX CH3 and Gal IV H2. The latter

rOes are similar to those seen for the Le
a
epitope (see
above), as the Le
x
epitope also shows stacking interaction
between the Fuc IX and Gal IV residues, resulting from very
similar conformational features ([21] and references therein).
Gal IV on the 6-branch is further extended at the 3-position,
as GlcNAc V shows an rOe to H3 of Gal IV. GlcNAc V is
substituted at the 3- and 4-position, as Gal VI H1 gives an
rOe to GlcNAc V H3 and to the NAc protons of GlcNAc
V, while Fuc X H1 gives an rOe to GlcNAc V H4. This
second Le
a
epitope (see also 3-branch above) also shows
remote rOes between H5 and CH3 of Fuc X and H2 of
Gal VI.
The TFiLNO structural assignment is further supported
by similar chemical shifts observed for the protons of
residues on the 6-branch to those previously reported for an
iso-lacto-N-octaose that contains this difucosylation on the
6-branch [17].
Thus, TFiLNO has the following structure:
MFiLNO. The monosaccharide composition of MFiLNO
was shown by methylation analysis to consist of four Gal
residues, three GlcNAc residues, one reducing Glc residue,
and one fucose residue (see above).
1
H chemical-shift

assignments of these residues (Table 4) were made from
TOCSY spectra. The Gal and GlcNAc residues are involved
Table 2.
1
H chemical shifts, H
1
,H
2
coupling constants, and intermolecular rOes from NMR spectra of iLNO. Chemical shifts from a 1D
1
H 800-MHz
spectrum recorded at 15 °C are given to three decimals. Other chemical shifts were taken from 2D spectra.
Residue Linkages
Chemical shifts in p.p.m. and (H1,H2 coupling constants in Hz)
rOes (from H1)
H1 H2 H3 H4 H5 H6a/b NAc
I Glca 5.22 (3.6) 3.59
I Glcb 4.665 (8.0) 3.292 3.63 3.61 3.61 3.944/3.80
II Gal 4 4.427(8.0) 3.58 3.73 4.149 3.84 3.99/3.84 3.61(H4,Ib)
III GlcNAc 6,4 4.652a 3.75 3.72 3.72 3.62 3.99/3.84 2.058 3.84(H6b,II);
4.634b(8.0) 3.99(H6a,II)
IV Gal 4,6,4 4.47(7.9) 3.58 3.74 4.158 3.72 3.75 3.72(H3,H5
a
; H4III)
V GlcNAc 3,4,6,4 4.71(8.3) 3.91 3.82 3.58 3.48 3.91/3.80 2.027 3.74(H3II,IV)
VII GlcNAc 3,4
VI Gal 3,3,4,6,4 4.45(8.0) 3.523 3.642 3.91 3.71 3.82(H3V,VII)
VIII Gal 3,3,4 2.02(NAc,V,VII)
a
Intramolecular rOes originating from Gal IV H1.

Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1181
in b-anomeric linkages as deduced from H
1
,H
2
coupling
constants between 7.7 and 8.1 Hz, while the fucose residue is
a-linked to its neighbouring residue, deduced from the
H
1
,H
2
coupling constant of 3.7 Hz (Table 4).
The sequence of the oligosaccharide was derived from
interresidue rOes (Table 4) and fully confirmed the MS
assignment. The reducing Glc residue is substituted at
position 4, as Gal II H1 of the b-anomer shows an rOe to
H4 of this residue. Gal II is a branching point residue and
substituted at positions 3 and 6, as GlcNAc III H1 gives
rOes to H6b of Gal II, while GlcNAc VII H1 gives an rOe to
H3 of Gal II. The 3-branch is further extended by a terminal
Gal VIII, as Gal VIII H1 gives an rOe to GlcNAc VII H3.
The GlcNAc III on the 6-branch is substituted at the
3- and 4-position, as Gal IV H1 gives an rOe to H4 of
GlcNAc III, and Fuc IX H1 gives an rOe to H3 and to the
NAc protons of this residue. The trisaccharide Le
x
epitope,
Gal IVb1–4(Fuc IXa1–3)GlcNAcIII, is also characterized
by rOes between Fuc IX H5 and Gal IV H2 and between

Fuc IX CH3 and Gal IV H2 (see also TFiLNO above). Gal
IV is substituted at the 3-position, as GlcNAc V H1 gives an
rOe to Gal IV H3, and the GlcNAc V residue is substituted
by a terminal Gal VI residue, as Gal VI H1 gives an rOe to
GlcNAc V H3.
The MFiLNO assignment is further supported by
chemical-shift comparisons with the nonfucosylated iLNO.
The two oligosaccharides have very similar chemical shifts
for the reducing Gal IIb1–4Glc residues, for the 3-branch
and for the GalVIb1–3GlcNAcV residues. Chemical shifts
for the residues forming the Le
x
epitope are very similar to
those residues in the TFiLNO structure.
The structure of MFiLNO is thus assigned as:
DFiLNO. The methylation analysis for DFiLNO identi-
fied a decasaccharide. The chemical shifts of anomeric
protons of the reducing terminal Glc at 5.216 and
4.67 p.p.m. for a and b anomers, respectively, four Gal
residues at 4.42 (II), 4.43 (IV), 4.515 (VI) and 4.44
(VIII) p.p.m., three GlcNAc residues at 4.632 (III), 4.674
(V) and 4.717 (VII), and two Fuc residues at 5.094 (IX) and
5.035 (X) (data not shown), are very similar to those
reported previously for DFiLNO [17,22]. Hence the
structure of the main component is as shown below and
in accord with the MS assignment. A minor component (see
above) is not apparent from the NMR spectrum because of
overlapping chemical shifts with the major component.
Table 3.
1

H chemical shifts, H
1
,H
2
coupling constants, and intermolecular rOes from NMR spectra of TFiLNO. Chemical shifts from a 1D
1
H 600-
MHz spectrum recorded at 27 °C are given to three decimals. Other chemical shifts were taken from 2D spectra.
Residue Linkages
Chemical shifts in p.p.m. and (H1,H2 coupling constants in Hz)
rOes (from H1)
H1 H2 H3 H4 H5 H6a/b NAc
I Glca 5.218(3.7) 3.59
I Glcb 4.665(8.0) 3.287 3.65 3.61 3.61 3.94/3.80
II Gal 4 4.427(7.8) 3.58 3.71 4.138 3.83 3.98/3.83 3.61(H4b,I)
III GlcNAc 6,4 4.639(7.8) 3.91 3.91 3.91 3.60 2.049 3.83(H6b,II)
IV Gal 4,6,4 4.433(8.0) 3.52 3.71 4.098 3.59 3.71 3.91(H4III)
H4
a
:3.59
(H5),
3.71(H6a, 6b)
V GlcNAc 3,4,6,4 4.697(8.4) 3.96 4.08 3.77 3.55 3.88 2.030 3.71(H3II,IV)
VII GlcNAc 3,4
VI Gal 3,3,4,6,4 4.515/4.503 3.49 3.620 3.89 3.58 4.08(H3V,VII)
VIII Gal 3,3,4 (7.8) 3.96(H2V,VII)
2.03(NAc, V,VII)
IX Fuc 3,6,4 5.090(3.8) 3.70 3.88 3.775 4.806 1.150 3.91(H3III)
2.05(NAc III)
H5 : 3.52

(H2IV)
CH
3
: 3.52
(H2IV)
X Fuc 4,3,4,6,4 5.030(3.8) 3.80 3.88 3.79 4.873 1.180 3.77(H4V,VII)
XI Fuc 4,3,4 H5 : 3.49
(H2VI, VIII)
CH
3
: 3.49
(H2VI, VIII)
a
Intramolecular rOes originating from Gal IV H4.
1182 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
We have isolated various fucosylated neutral oligosaccha-
rides from human milk that are based on the iso-lacto-
N-octaose core, and fully characterized four structures by
the combined use of ES-MS, methylation analysis and
NMR spectroscopy. The branching pattern and blood
group-related Lewis determinants can be readily deter-
mined by high-sensitivity ES-MS/MS analysis as demon-
strated here and also previously [13,14]. Even a minor
isomeric component present in a mixture can be detected,
as exemplified by the analysis of DFiLNO. NMR is
currently the only technique that can provide the full
sequence information, including linkage and a,b-anor-
meric configuration. However, because of the complexity
of overlapping signals of the same monosaccharide

residues in a very similar chemical environment, it is
difficult for NMR alone to assign the branching pattern.
The combined use of product-ion scanning of negative-ion
ES-MS and NMR has proved a powerful strategy for
complete assignment of the branched structures. NMR
Fig. 7. 1D and 2D
1
H-NMRspectra(600MHz)ofTFiLNOat27°C. Upper trace, the 5.5–0.5 p.p.m. region of the
1
H-NMR spectrum; bottom
trace, the 5.5–0.5 by 2.5–0.5 p.p.m. regions of the 300-ms ROESY spectrum. The structure is shown to indicate the residue labelling.
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1183
chemical shifts have been assigned for all compounds and
this opens the way to their conformational analysis by
NMR.
Several variously fucosylated neutral iso-lacto-N-octaose
derivatives have been described previously, including two
difucosylated [17], one trifucosylated [23], one tetrafucosyl-
ated [24], and one pentafucosylated [24] oligosaccharides.
Acidic structures described include one monosialyl-mono-
fucosylated [25], two monosialyl-difucosylated [25] and one
monosialyl-trifucosylated [26] oligosaccharides. In the pre-
sent study, three structures have been isolated for the first
time. The nonfucosylated iso-lacto-N-octaose has not been
Fig. 8. 1D and 2D
1
H-NMR spectra (600 MHz) of TFiLNO, region 5.5–3.0 p.p.m., at 27 °C. Upper trace, 1D
1
H spectrum; Top-left half, 300-ms
ROESY spectrum and bottom-right half, 140-ms TOCSY spectrum. The structure is shown to indicate the residue labelling.

1184 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
reported as an individual oligosaccharide, although it has
been described previously as a core type [24] based on the
identification of its fucosylated derivatives. The mono-
fucosylated iso-lacto-N-octaose, containing a Fuc at the first
GlcNAc residue of the 6-branch forming an internal Le
x
epitope, and the trifucosylated iso-lacto-N-octaose, contain-
ing two terminal Le
a
epitopes at each of the branches and
one internal Le
x
on the 6-branch are also novel structures.
Acknowledgements
We thank Dr Colin Herbert for his technical support. This work
was supported by a UK Medical Research Council programme grant
(G9601454). NMR experiments and analysis were carried out at the
MRC Biomedical NMR centre, National Institute of Medical
Research, London.
References
1. Gottschalk, A. (1954) The influenza virus enzyme and its muco-
protein substrate. Yale J. Biol. Med. 26, 352–364.
2. Watkins, W.M. (1972) Glycoproteins: their composition, structure
and function. In Blood-Group Specific Substances (Gottschalk, A.,
ed.), pp. 830–899. Elsevier, Amsterdam.
3. Kabat, E.A. (1982) Contributions of quantitative immunochem-
istry to knowledge of blood group A, B, H, Le, I and i antigens.
Am. J. Clin. Pathol. 78, 281–292.
4. Feizi, T. (2000) Carbohydrate-mediated recognition systems in

innate immunity. Immunol. Rev. 173, 79–88.
5. Feizi, T. (1985) Demonstration by monoclonal antibodies that
carbohydrate structures of glycoproteins and glycolipids are onco-
developmental antigens. Nature 314, 53–57.
6. Newburg, D.S. & Neubauer, S.H. (1995) Carbohydrates in milks:
analysis, quantities and significance. Handbook of Milk Composi-
tion, pp. 273–349. Academic Press, San Diego.
7. Yamashita, K., Mizuochi, T. & Kobata, A. (1982) Analysis of
oligosaccharides by gel filtration. Methods Enzymol. 83, 105–126.
8. Kunz,C.,Rudloff,S.,Baier,W.,Klein,N.&Strobel,S.(2000)
Oligosaccharides in human milk: structural, functional, and
metabolic aspects. Annu. Rev. Nutr. 20, 699–722.
9. Rudloff, S., Stefan, C., Pohlentz, G. & Kunz, C. (2002) Detection
of ligands for selectins in the oligosaccharide fraction of human
milk. Eur. J. Nutr. 41, 85–92.
10. Martin, M.J., Feizi, T., Leteux, C., Pavlovic, D., Piskarev, V.E. &
Chai, W. (2002) An investigation of the interactions of E-selectin
with fuco-oligosaccharides of the blood group family. Glycobio-
logy 12, 829–835.
11. DeFrees, S.A., Kosch, W., Way, W., Paulson, J.C., Sabesan, S.,
Halcomb,R.L.,Huang,D.H.,Ichikawa,Y.&Wong,C.H.(1995)
Ligand recognition by E-selectin: synthesis, inhibitory activity,
and conformational analysis of bivalent sialyl Lewis x analogs.
J. Am. Chem. Soc. 117, 66–79.
12. Chai, W., Feizi, T., Yuen, C T. & Lawson, A.M. (1997) Non-
reductive release of O-linked oligosaccharides from mucin glyco-
proteins for structure/function assignments as neoglycolipids:
application in the detection of novel ligands for E-selectin.
Glycobiology 7, 861–872.
13. Chai, W., Piskarev, V. & Lawson, A.M. (2001) Negative-ion

electrospray mass spectrometry of neutral underivatized oligo-
saccharides. Anal. Chem. 73, 651–657.
14. Chai, W., Piskarev, V. & Lawson, A.M. (2002) Branching pattern
and sequence analysis of underivatized oligosaccharides by com-
bined MS/MS of singly and doubly charged molecular ions in
negative-ion electrospray mass spectrometry. J. Am. Soc. Mass
Spectrom. 13, 670–679.
15. Domon, B. & Costello, C.E. (1988) A systematic nomenclature for
carbohydrate fragmentations in FAB-MS/MS spectra of glyco-
conjugates. Glycoconjugate J. 5, 397–409.
16. Chai, W., Hounsell, E.F., Cashmore, G.C., Rosankiewicz, J.R.,
Bauer,C.J.,Feeney,J.,Feizi,T.&Lawson,A.M.(1992)Neutral
oligosaccharides of bovine submaxillary mucin: a combined mass
spectrometry and
1
H-NMR study. Eur. J. Biochem. 203, 257–268.
17. Strecker, G., Wieruszeski, J.M., Michalski, J.C. & Montreuil, J.
(1989) Primary structure of human milk nona- and decasacchar-
ides determined by a combination of fast atom bombardment
Table 4.
1
Hchemicalshifts,H
1
,H
2
coupling constants, and intermolecular rOes from NMR spectra of MFiLNO IV. Chemical shifts from a 1D
1
H
500-MHz spectrum recorded at 10 °C are given to three decimals. Other chemical shifts were taken from 2D spectra.
Residue Linkages

Chemical shifts in p.p.m. and (H1,H2 coupling constants in Hz)
rOes (from H1)
H1 H2 H3 H4 H5 H6a/b NAc
I Glca 5.214(3.9) 3.60
I Glcb 4.663(8.1) 3.285 3.64 3.61 3.64 3.95
II Gal 4 4.43(7.8) 3.57 3.72 4.15 3.82 3.99/3.84 3.61(H4b,I)
H4
a
:3.99,3.84
(H6a,H6b)
III GlcNAc 6,4 4.64(7.7) 3.90 3.90 3.90 2.049 3.84(H6b,II)
IV Gal 4,6,4 4.44(7.8) 3.53 3.72 4.11 3.58 3.90(H4III)
2.02(NAc,V)
V GlcNAc 3,4,6,4 4.708(7.8) 3.90 3.80 3.59 3.48 2.022 3.72(H3II, IV)
VII GlcNAc 3,4
VI Gal 3,3,4,6,4 4.45(7.8) 3.51 3.64 3.91 3.80(H3V, VII)
VIII Gal 3,3,4
IX Fuc 3,6,4 5.091(3.7) 3.68 3.88 3.77 4.824 1.145 3.90(H3III)
2.05(NAc,III)
H5 : 3.53(H2, IV)
CH
3
: 3.53(H2IV)
a
Intramolecular rOes originating from Gal II H4.
Ó FEBS 2004 Sequence determination of oligosaccharides (Eur. J. Biochem. 271) 1185
mass spectrometry and
1
H-/
13

C-nuclear magnetic resonance
spectroscopy. Evidence for a new core structure, iso-lacto-
N-octaose. Glycoconj. J. 6, 169–182.
18. Dua, V.K., Goso, K., Dube, V.E. & Bush, C.A. (1985)
Characterization of lacto-N-hexaose and two fucosylated deriva-
tives from human milk by high-performance liquid chromato-
graphy and proton NMR spectroscopy. J. Chromatogr. 328,
259–269.
19. Lemieux, R., Bock, K., Delbaere, L., Koto, S. & Rao, V. (1980) The
conformations of oligosaccharides related to the ABH and Lewis
human blood group determinants. Can. J. Chem. 58, 631–653.
20.Bechtel,B.,Wand,A.J.,Wroblewski,K.,Koprowski,H.&
Thurin, J. (1990) Conformational analysis of the tumor-associated
carbohydrate antigen 19–9 and its Le
a
blood group antigen
component as related to the specificity of monoclonal antibody
CO19-9. J. Biol. Chem. 265, 2028–2037.
21. Kogelberg, H., Frenkiel, T.A., Homans, S.W., Lubineau, A. &
Feizi, T. (1996) Conformational studies on the selectin and natural
killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses
(SuLNFPII and SLNFPII) using NMR spectroscopy and
molecular dynamics simulations. Comparisons with the nonacidic
parent molecule LNFPII. Biochemistry 35, 1954–1964.
22. Sabharwal, H., Nilsson, B., Gronberg, G., Chester, M.A.,
Dakour, J., Sjoblad, S. & Lundblad, A. (1988) Oligosaccharides
from feces of preterm infants fed on breast milk. Arch. Biochem.
Biophys. 265, 390–406.
23. Strecker, G., Fievre, S., Wieruszeski, J.M., Michalski, J.C. &
Montreuil, J. (1992) Primary structure of four human milk octa-,

nona-, and undeca-saccharides established by
1
H- and
13
C-nuclear
magnetic resonance spectroscopy. Carbohydr. Res. 226, 1–14.
24. Haeuw-Fievre, S., Wieruszeski, J.M., Plancke, Y., Michalski, J.C.,
Montreuil, J. & Strecker, G. (1993) Primary structure of human
milk octa-, dodeca- and tridecasaccharides determined by a
combination of
1
H-NMR spectroscopy and fast-atom-bombard-
ment mass spectrometry. Evidence for a new core structure, the
para-lacto-N-octaose. Eur. J. Biochem. 215, 361–371.
25. Kitagawa, H., Nakada, H., Fukui, S., Funakoshi, I., Kawasaki,
T., Yamashina, I., Tate, S. & Inagaki, F. (1991) Novel oligo-
saccharides with the sialyl-Le
a
structure in human milk. Bio-
chemistry 30, 2869–2876.
26. Kitagawa, H., Nakada, H., Fukui, S., Funakoshi, I., Kawasaki,
T., Yamashina, I., Tate, S. & Inagaki, F. (1993) Novel oligo-
saccharides with the sialyl-Le (a) structure in human milk.
J. Biochem. (Tokyo) 114, 504–508.
1186 H. Kogelberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004