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

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-

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

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

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

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

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

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

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

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- – – – –

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

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

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

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

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

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

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

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.

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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

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H- and
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