Glycobiology vol. 9 no. 3 pp.

255266, 1999

Gas-liquid chromatography of the heptafluorobutyrate derivatives of the

O-methyl-glycosides on capillary columns: a method for the quantitative
determination of the monosaccharide composition of glycoproteins and glycolipids

Jean-Pierre Zanetta1, Philippe Timmerman and

Yves Leroy
Laboratoire de Chimie Biologique, CNRS UMR 111, 59655 Villeneuve
dAscq Cedex, France
Received on May 18, 1998; revised on July 22, 1998; accepted on July 27,
1998
1To

whom correspondence should be addressed at: Laboratoire de Chimie

Biologique USTL, CNRS UMR 111, 59655 Villeneuve dAscq Cedex,
France

We have developed a method involving the formation of heptafluorobutyrate derivatives of O-methyl-glycosides liberated
from glycoproteins and glycolipids following methanolysis.
The stable derivatives of the most common monosaccharides
of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc,
GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and
quantitatively and reproducibly determined with a high
degree of sensitivity level (down to 25 pmol) in the presence
of lysine as an internal standard. The GlcNAc residue bound
to Asn in N-glycans is quantitatively recovered as two peaks.
The latter were easily distinguished from the other GlcNAc
residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a
single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of
soluble or membrane-bound glycoproteins (SDS, Triton
X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as
well as monosaccharide constituents of proteoglycans or
degradation products of nucleic acids) do not interfere with
these determinations. A carbohydrate analysis of glycoproteins
isolated from a SDS/PAGE gel or from PDVF membranes can
be performed on microgram amounts without significant
interferences. Since fatty acid methyl esters and sphingosine
derivatives are separated from the monosaccharide peaks,
the complete composition of gangliosides can be achieved in
a single step starting from less than 1 g of the initial compound purified by preparative Silicagel TLC. Using electron
impact ionization mass spectrometry, reporter ions for the
different classes of O-methyl-glycosides (pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, sialic acid, and
KDN) allow the identification of these compounds in very
complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive
or negative ions. This method presents a considerable
improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and
acylation of amino groups is complete. Moreover, there is no
interference with contaminants and the separation between
fatty acid methyl-esters and O-methyl glycosides is achieved.
Key words: GLC/GC/MS, mass spectrometry/monosaccharide
1999 Oxford University Press

Introduction
The interest over the carbohydrate moieties of glycoconjugates has
been increasing because of their postulated functions in cell adhesion
and recognition mechanisms and their importance in signal
transduction and as markers of differentiation and carcinogenesis.
The determination of the monosaccharide composition is a key step
in choosing the strategy for glycan analysis. The most common
method is the capillary column GLC analysis of the trimethylsilyl
derivatives (TMS) obtained from O-methyl-glycosides after methanolysis induced cleavage of glycosidic bonds (for review, see
Chaplin, 1994). This cleavage procedure, when used in controlled
conditions (and especially the use of anhydrous methanol/HCl
reagent; Zanetta et al., 1972), allows the almost quantitative
liberation of the glycoprotein and glycolipid monosaccharides as
stable O-methyl-glycosides. However, an intermediate step of
re-acetylation of these compounds is necessary before the formation
of the TMS derivatives in the classical procedure (for review, see
Chaplin, 1994). Unfortunately, this re-acetylation step is time
consuming, and the reaction yield is particularly difficult to control.
As a result, the molar ratio of N-acetyl-hexosamines and sialic acid
fluctuates from an experiment to the other, rendering monosaccharide molar composition of a glycan often problematic.
One way to circumvent such a difficulty is the formation of
volatile derivatives of O-methyl-glycosides using a strong
acylating agent (acid anhydride). In a previous paper (Zanetta et al.,
1972), the most common constituents of glycoproteins and
glycolipids (and all constituents of glycolipids) could be determined
quantitatively as trifluoroacetate derivatives (TFA) using a
separation by classical GLC. However, this method lacked the
sensitivity required for applications to small amounts of material
(less than 100 g of glycoprotein). The reason was that the use of
capillary columns was prevented by the presence of an excess of
trifluoroacetic anhydride which destroyed the liquid phase.
Because of the volatile nature of the TFA derivatives, the excess
of anhydride could not be eliminated without the complete loss
of the most volatile compounds. We hypothesized that one way
to solve this problem was to produce less volatile derivatives
using an acylation with anhydrides of higher mass (pentafluoropropionic or heptafluorobutyric anhydrides). This article demonstrates that quantitative monosaccharide analyses of glycoproteins
and glycolipids can be achieved on capillary columns using the
heptafluorobutyrate derivatives of the O-methyl-glycosides.
Results and discussion
Choice of the acylating agent
Standard monosaccharide mixtures were submitted to methanolysis
(see Materials and methods), followed by acylation with
pentafluoropropionic or heptafluorobutyric anhydrides, respectively. The derivatives were injected into the SE-30 column, and
the area of the different peaks was quantified. These areas were
compared to those of the same compounds injected after the
complete evaporation of the acylation mixture under a stream of
255

J.-P.Zanetta, P.Timmerman and Y.Leroy

nitrogen and resolubilization in dried acetonitrile. Pentafluoropropionate derivatives of fucose, arabinose, and xylose and to a
lesser extent the hexose derivatives showed a significant decrease
during evaporation (a maximum of 20% loss for the most volatile
compounds). In contrast, identical areas were found for all
heptafluorobutyrate derivatives, even working with less than
1 nmol of each monosaccharide in the reaction vessel. This
adequate volatility was verified after injection on capillary
columns, the surface of the peaks being constant after evaporation
of the solvent in the Ross injector.
Conditions of acylation
The acylation reaction with HFBAA was performed in acetonitrile
because this solvent was able to solubilize the O-methyl-glycosides formed during methanolysis and because it formed a single
phase with HBFAA (in contrast with less polar solvents such as
dichloromethane, chloroform, heptane, methyl or ethyl acetate).
Kinetics of acylation of the different O-methyl-glycosides were
followed either at room temperature or at 100
C or 150
C,
analysing the areas of the different peaks of standard mixtures.
When experiments were performed at room temperature, the
acylation was complete only after 24 h, the acylation being slower
for monosaccharides possessing amino groups (hexosamines and
sialic acid). When acylation was performed at 100
C, the
acylation was maximal after 30 min; the relative molar responses
did not vary for longer times of heating. At 150
C, the acylation
was complete within 5 min (this procedure being recommended
since the reaction takes place as a reflux, also acylating the
material remaining on the vessel wall).
Because acetonitrile showed some trailing on classical columns,
we tried to solubilize the evaporated HFB derivatives in other
solvents, i.e., heptane or dichloromethane. Heptane provoked an
important loss of most compounds (especially hexosamines and
sialic acid, but also pentoses and hexoses) at all initial concentrations of monosaccharides because HFB derivatives were not
soluble. A significant loss of the same compounds was observed
with dichloromethane when the initial concentration of the
monosaccharide was more than 5 nmol in the vessels. In contrast,
acetonitrile completely dissolved the different compounds and
appeared suitable for capillary column analysis.
Stability of the heptafluorobutyrate derivatives
After acylation, the HFB-derivatives kept at room temperature in the
closed reaction vial were analyzed at different periods of time. No
variations were observed during several months, as already observed
for the trifluoroacetate derivatives (Zanetta et al., 1972). The same
type of experiment was also performed on the HFB derivatives

stored for different periods of times in dried acetonitrile after the

complete evaporation of the acylation mixture. No variations were
observed when samples were stored for 2 days in the closed vial with
the exception of the derivatives corresponding to the GlcNAc
residue involved in the N-glycosidic bond (see below). Repetitive
(up to 10 times) opening of the vials for time to time injections did
not modify significantly the relative molar responses of the different
compounds with the exception of the previous derivatives and,
specifically, the derivatives of the furanic forms of all compounds
(Fuc, Gal, GalNAc, GlcA, and GalA) and the derivatives of sialic
acid and KDN. However, the initial response of these compounds
could be restored, adding 25 l of HFBAA and heating for 5 min
at 100
C. This indicated that the HFB derivatives presented a strong
stability in neutral or acidic conditions. Because of this stability, the
cleaning of the reaction vessels before reuse should first involve an
immersion in an alkaline solution (1 M NaOH for 10 min). Tubes
were cleaned thereafter by heating for 1 h at 80
C in 50% sulfuric
acid.
Choice of GLC separations
From previous observations (Zanetta et al., 1972, 1973), it appeared
that fluorinated compounds strongly interact with fluorosilicone
liquid phases and are poorly adsorbed on classical silicone phases,
in such a way that they are eluted from the columns at temperature
lower than their boiling points. In contrast, compounds rich in
methylene groups strongly interact with classical silicone phases. In
fact, despite their high mass, HFB of O-methyl-glycosides were
eluted before fatty acid methyl-esters (FAMEs).
The chosen chromatographic conditions were: injector and
detector temperature 260
C; helium carrier gas 0.8 bar;
temperature program: 100
C to 140
C at 1.2
C/min, followed
by 4
C/min until 240
C (when samples contained only monosaccharide derivatives, the temperature program was stopped
after the elution of the neuraminic acid peak). Using these
conditions, almost all HFB of the isomers of O-methyl-glycosides (Xyl, Ara, Rha, Fuc, Gal, Man, Glc, GlcNAc, GalNAc,
NeuAc, and KDN) were separated from each other (Figure 1,
Table I). The single overlapping was observed for the -mannose
isomer and a furanic form of GalNAc. Furthermore, FAMEs were
eluted at higher retention time than the derivatives of NeuAc. The
quality of the separations remained stable during at least six
months of intense use (about 600 injections), indicating that the
derivatives did not destroyed the columns. However, when HFB
derivatives are chromatographed on a column on which other
derivatives were analyzed (acetates or TMS derivatives), the
separation of the second isomer of Glc and the third of Gal are
only partially achieved. This partial overlapping disappeared
after two to three runs of HFB derivatives.

Fig. 1. Gas chromatography of the heptafluorobutyrates of O-methyl glycosides on a capillary column. (a) GC chromatogram of a mixture of standard
monosaccharides (Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac) and fatty acids (C16, C18, C20, C22 as major compounds) submitted to methanolysis then
acylation by HFBAA. Note that all constituents of N-glycans are separated the one from the other and completely separated from FAME. The -isomer of Glc is
only partially resolved from a galactose isomer due to the injection of HFB derivatives immediately after a chromatography of TMS derivatives. Inset: separation
of the different isomers of O-methyl glycosides of pentoses and deoxy-hexoses (Ara, Rha, Xyl, and Rib). The lines inside the chromatogram represent the
temperature program. (b) GC chromatogram of glycoproteins found in wasps net. Note the complete separation of Ara, Rha, Fuc, the presence of two peaks
(GN1) corresponding to the first GlcNAc residue of N-glycans. This chromatogram revealed the presence of small amounts of glucuronic acid (peak in-between
the major peaks of Gal and Man, and the two peaks migrating between the two first peaks of GalNAc; Rt = 25.43, 31.95, and 32.39 min, respectively). (c) GC
chromatogram of the mucins of the eggs of Pleurodeles walti. This materiel contained high amounts of KDN, perfectly separated from all other compounds. The
carbohydrate composition deduced from this analysis performed starting from 10 g of mucin was identical (less than 1% variation) to a previous analysis
performed on 1 mg mucin using the TFA derivatives (Zanetta et al., 1972). Because these material were injected onto a column perturbed by the injection of
acetylated compounds, the separation of the Glc and Gal isomers was not optimal.

256

HFB derivatives of O-methyl glycosides

257

J.-P.Zanetta, P.Timmerman and Y.Leroy

Table I. Retention time and proportions of the different isomers of the
heptafluorobutyrates of O-methyl-glycosides (the Rt are reproducible within
0.02% of min)
Compounds
Meso
Manni
Xyl
Xyl
Ara
Ara
Rha
Rha
Rib
Rib
Rib
Fuc
Fuc
Fuc
Fuc
Gal
Gal
Gal
Gal
Man
Man
Glc
Glc
GlcNa
GlcNa
GlcN
GlcN
GlcN
GalN
GalN
GalN
GalN
Neu5
Neu5
KDN
KDN
GlcN-OH
GalN-OH
GlcA
GlcA
GlcA
GalA
GalA
GalA
GalA
ManNb
ManNb
ManNb
Asn
Lys
aThe

Retention time
23.09
15.19
16.01
16.32
11.75
14.91
14.55
17.70
13.83
16.83
18.66
13.43
14.43
16.73
18.94
20.04
22.79
25.37
27.46
26.41
29.49
26.89
27.27
20.17
22.29
34.98
35.90
36.56
29.41
32.65
35.48
36.72
42.21
43.80
38.97
39.72
16.32
20.63
25.43
31.95
32.39
21.12
22.91
31.51
33.61
34.64
35.77
36.85
11.09
38.65

% of total
100.00
100.00
66.48
33.52
16.82
83.18
94.48
5.52
14.76
15.38
69.86
12.04
5.96
55.23
26.67
19.75
6.18
49.38
24.69
93.05
6.95
67.49
32.51
48.90
51.10
6.67
83.83
9.50
18.13
24.30
52.59
4.98
11.83
88.17
27.78
72.22
100.00
100.00
29.63
17.32
53.05
41.71
13.76
36.46
8.07
34.49
42.69
19.37
100.00
100.00

two peaks correspond to the derivatives formed during the methanolysis

of the glycosylamine of GlcNAc involved in the N-glycosidic bond ( and
isomers of glucosamine).
bThe derivatives of N-acetyl-mannosamine interfere with the separation of
those of GlcNAc and GalNAc, but this compound has not been so far
identified in glycoproteins or glycolipids.

258

Choice of an appropriate internal standard

Classical techniques used polyols as internal standard (such as
meso-inositol or mannitol). In fact mannitol could be released by
reductive -elimination procedure from glycans with an O-linked
mannose residue (Chiba et al., 1997), whereas meso-inositol is an
essential constituent of GPI-anchors. Moreover, in the present
system, meso-inositol showed trailing and interfered with the
second Gal isomer. Because of these possible interferences, we
preferred amino acids, the carboxyl group being methyl-esterified
during methanolysis and the amino group acylated with HFBAA.
Throughout the different amino acids presenting stable heptafluorobutyrate derivatives (Zanetta and Vincendon, 1973), lysine
(Rt = 38.65) showed a chromatographic behavior compatible
with the separation of the classical monosaccharides of glycoproteins and of glycolipids and did not interfere with any
monosaccharides. Because of the presence of two minor impurities
in commercially available lysine, it was necessary to recrystallize
it from water. Subsequent studies of the possible contaminations
of glycolipid and glycoprotein samples (see below) indicated that
lysine was a suitable internal standard.

Determinations of the relative molar responses

The relative molar responses (RMR) of the different HFB
derivatives were first determined on equimolar mixtures of
different monosaccharides submitted to methanolysis in the
standard conditions, followed by acylation with HFBAA and
analyzed on the two types of GLC columns. Because of the
presence of significant amounts of impurities in commercially
available standards, these RMR were only approximate. Corrections
due to the presence of the impurities eliminated some divergences
observed between the different compounds of the same mass.
These RMR were compared to those obtained from the analysis
of oligosaccharides, reduced oligosaccharides or glycoasparagines, the structure and purity has been previously determined by
methylation and NMR analysis. The final data from these
determinations are presented in Table II: (1) taking into accounts
all isomers of the different monosaccharides (RMR); (2) taking
into account the major isomer (RMRmp), since the proportions
of the different isomers of each monosaccharides were constant
(with the exception of the derivative of sialic acid and that of
galactose). Therefore, the quantitation of an analysis could be
performed into two steps: (1) verification that the proportions of
the different isomers are correct (a point that indicated the
absence of contaminant in the major peaks); (2) calculation of the
area of each monosaccharide based on the RMR of the major peak
(RMRmp; Table II). The late form of calculation could not be
applied systematically to the NeuNAc derivatives because theses
compound are eluted, depending on the sample, either as a single
peak or as two peaks. In the latter case, the major anomer
represented always 88.17%, a point that remains to be explained.
Similarly, the integration of the two peaks corresponding to the
derivatives of the major product issued from the GlcNAc residue
involved in the N-glycosidic bond, i.e., glucosamine (Maes et al.,
1999), was needed, since these proportions were varying with
time, due to a slow change in the proportions of the and
isomers from a methanol/HCl to an acetonitrile medium. The case
of the response of Gal will be discussed below.

HFB derivatives of O-methyl glycosides

Table II. Relative molar response (RMR) of the heptafluorobutyrates of
O-methyl-glycosides and relative molar response relative of the major isomer
of each compound (RMRmp)
Compounds
Meso
GalNac-OH
GlcNac-OH
Xyl
Ara
Rha
Fuc
Gal
Man
Glc
GlcNAc
GalNAc
Neu5Ac
KDN
GlcAd
GalAd
GlcNc
Asn
2Lys (IS)

RMR/IS
(all peaks integrated)
1.000
0.850
0.850
0.850
0.750
0.900
0.896
0.755e
0.930
0.930
0.872
0.751
1.000
1.000
0.950
0.930
0.750
0.500
1.000

RMRmp/IS (only the

major peak integrated)
1.000
0.850
0.850
0.565
0.624
0.850
0.495
0.559e
0.865
0.628
0.731
0.395
0.882b
0.722a
0.504
0.388
0.500
1.000

The RMR (0.2% of the indicated values) are mean of at least 30 different
determinations both on standard of monosaccharides submitted to
methanolysis then acylation with HFBAA and on glycoproteins, glycolipids
or oligosaccharides and glycoasparagines of known compositions.
aTheoretical value, deduced from the RMR of the derivatives of Neu5Ac.
bIn some glycoconjugates and on the standard Neu5Ac, two peaks were
observed; the RMR = 0.882 correspond to that of the major peak of NeuAc.
In analyses where Neu5Ac gave a single peak, the RMR = 1.000 of NeuNAc
was applied.
cBecause of the variations in the relative proportions of the two peaks, the
RMR correspond to the result of the sum of their area.
dNot released significantly from glycosaminoglycans during methanolysis
(115% depending on the compounds), but could be partially released from
branched oligosaccharides. For simplifying the calculations of the
carbohydrate content of glycoproteins or glycolipids, the addition of 2 g of
Lys as the internal standard was considered as 1 g of internal standard (IS),
since the relative molar response of Lys is only half of that of many O-methyl
glycosides.
eWhen O-glycans are present (presence of GalNAc or GalNAc-OH, the
surface of all galactose peaks should be integrated since the proportion of the
first isomer (Rt = 20.04) is significantly increased. The RMRmp allows a
rapid calculation of the molar ratio since calculation is based only on the area
of the major isomer of each monosaccharide.

The analysis of the different glycoasparagines and of glycoproteins of known monosaccharide compositions indicated a
deficit of GlcNAc, when compared to the oligosaccharides with
a single or two GlcNAc residues. However, two major extraneous
peaks were detected for glycoproteins containing N-glycans at
Rt = 20.17 and 22.29 min, respectively (see Figures 1c, 2). A third
extraneous peak was observed for glycoasparagines (Rt =
11.09 min), that corresponded to the di-methyl-ester N-heptafluorobutyrate of aspartic acid by EI/MS (357) and CI/MS (M + 18 = 375).
Since this compound was a little bit too volatile (Zanetta and
Vincendon, 1973), it could not be exactly quantified using the
evaporation needed for capillary column analysis. Without
evaporation, and taking into account a RMR of 0.500 for this
derivative of Asp, it could be calculated that it corresponded to
one residue of Asp per glycoasparagine. This indicated that the
N-glycosidic bond was almost quantitatively cleaved (Maes et al.,

1999). The nature of the products liberated from GlcNAc-Asn by

methanolysis will be reported in detail elsewhere (Maes et al.,
1999). From the analysis of different N-glycoproteins and
glycoasparagines, a RMR of 0.750 was assigned to the sum of
these two compounds. Therefore, for the analysis of glycoproteins
containing N-glycans, the ratio of the different monosaccharides
was calculated relative to these constituents representative of the
GlcNAc residue involved in the N-glycosidic bond.
Table III. Retention times of fatty acid methyl esters and of sphingosine
derivatives obtained after methanolysis and acylation with HFBAA
FAME
C16:0
C18:0
C18:1
C18:2
C20:0
C20:3
C20:4
C22:4
C22:6
C24:0

Retention
time
48.98
54.55
53.92
53.76
58.65
57.97
57.47
62.72
62.35
65.34

RMR/IS

Sphingosine

1.620
1.800
1.790
1.780
2.300
2.280
2.250
2.560
2.540
2.830

C18:1
C18:1
C20:1
C20:1
C20:1

Retention
time
45.81
48.13
58.45
57.43
58.89

RMR/ISa
1.560
1.560
1.900
1.900
1.900

A more complete study of the fatty acid methyl esters and of the HFB
derivatives of hydroxylated FAMEs will be published elsewhere.
aThe RMR of the different sphingosines were determined from the data
obtained on the GM1 gangliosides, assuming that the response was
proportional to the number of carbon atoms. For FAMEs and sphingosine a
difference of Rt of 0.20 min resulted in a complete separation of the two peaks.

Applications to glycoprotein and glycolipids

Complete composition of gangliosides. HFB derivatives of
O-methyl-glycosides were completely separated from FAMEs
and from the HFB derivatives of sphingosines on the capillary
column. The C14:0 FAME was eluted later than the NeuAc HFB
derivative. The different FAME were fully separated from each
and are eluted before 240
C (Figure 1a). Because the derivatives
of sphingosines presented a high mass, they were eluted in
between fatty acids (Figure 2). Therefore, it was possible to
determine simultaneously the carbohydrate, sphingosine and
fatty acid composition of gangliosides without interference with
each other. The compositions of the GM1 and G5b gangliosides
are given in Table III. The determination of the nature of the fatty
acid methyl esters was obtained by comparison with standard
FAMEs and GC/MS analysis. The nature of the heptafluorobutyrate
derivatives of sphingosines was given by GC/MS analysis
(Figure 2). Using EI ionization, the different bases gave
characteristic fragments at M = 459 for the C18:1, M = 487 for
the C20:1, M = 489 for the di-hydro C20:0 and M = 543 for the
C24:1 derivatives of the sphingosine bases. Additional information
was obtained by chemical ionization and detection of the positive
ions with major fragments corresponding to [M + 18 - 214]+ of
M = 691, M = 719, M = 721, and M = 775 for the previous
compounds. It should be noted that in G5b, two different C18:1
sphingosines and three C20:1 sphingosines of the same mass are
observed (Figure 2), corresponding to a partial degradation
during the methanolysis step (Pons et al., unpublished data). The
elution of the C:24 derivative implied a prolongation of the
temperature up to 260
C. These determinations were in perfect
agreement with the data obtained by the MALDI-TOF analysis of
259

J.-P.Zanetta, P.Timmerman and Y.Leroy

Fig. 2. Identification of FAMEs ([M+ NH4]+) and sphingosine bases ([M 214 + NH4]+) of G5b ganglioside by GC/MS in the chemical ionization mode
(Gas chromatography was performed with a temperature gradient of 10
C/min from 90
C to 240
C. Note the presence of two peaks corresponding to the C18:1
sphingosine and three peaks corresponding to the C20:1 sphingosine. Derivatives of monosaccharides are eluted in the first part of the chromatogram (not shown).

these compounds. Attempts were also performed using the

detection of negative ions formed during chemical ionization, a
detection that increased the sensitivity of detection of heptaflurobutyrate derivatives but did not allow the detection of FAME.
The ions obtained using this detection system for the HFB
derivatives of O-methyl glycosides and of sphingosines were either
[M]
or [M - 20]
, the loss of mass of 20 corresponding to the
loss of HF. (Figure 3)
The RMR of FAMEs and sphingosines calculated from these
experiments (Table IV) approached the theoretical RMR calculated from the number of carbon and hydrogen atoms of the
molecules. It should be stressed that except for the quantity of
glucose, the molar ratios for these two standard gangliosides were
exact with a precision less than 1%.
Monosaccharide composition of glycoproteins. The GLC profiles
of the monosaccharides constituents of glycoproteins are shown
in Figures 1b, 1c, 2 and 3, and examples of carbohydrate
compositions are presented in Table V. Since the HFB derivative
of the major compounds resulting from the glycosylamine of
260

GlcNAc involved in the N-glycosidic bond (glucosamine; Maes

et al., 1999) was also quantified, the calculation of the molar
composition of N-glycans was performed relative to these
compounds. A series of glycoasparagines isolated from the
microsomal fraction of the adult rat brain were analyzed, starting
from 1 g amount of each glycan. The expression of the different
results relative to the GlcNAc residue involved in the N-glycosidic bond allowed obtaining the composition of the different
oligomannosides without ambiguities. But, because of the
relative instability of the glucosamine derivative, the samples had
to be analyzed within 48 h after the evaporation of the acylation
mixture. As mentioned above, the peak corresponding to the
di-methyl ester HFB of Asp, resulting from the Asn involved in
the N-glycosidic bond of glycoasparagines, could not be determined
quantitatively since this compound was partially lost during the
evaporation of the acylation mixture and during the drying step
in the Ross injector. Nevertheless, the presence of this peak was
indicative that the initial compound was a glycoasparagine and
not a glycan attached to a larger peptide.

HFB derivatives of O-methyl glycosides

Table IV. Composition of gangliosides
Compound
GM1
G5b

Fuc
0.000
0.000

Gal
2.000
2.000

Man
0.000
0.000

Glc
2.200
1.324

GlcNAc
0.000
0.000

GalNac
1.000
1.000

NeuAc
1.000
2.002

FAME
1.000
1.005

% of total
GM1 Sphing
G5b Sphing

C18:1
0.00
7.91

C18:1
53.38
16.18

C20:1
0.00
4.45

C20:1
0.00
21.06

C20:1
20.60
50.40

C20:0
26.10
0.00

C22:1
0.00
0.00

C24:1a
0.00
0.00

C16:0
0.0
6.7

C18:0
90.0
72.6

C20:0
10.0
3.5

C22:0
0.0
8.6

C24:0
0.0
8.6

Other
0.0
0.0

% of total
GM1 FAME
G5b FAME

Sphing
1.005
1.011

aThe

C24:1 sphingosine was found in a neutral lipid fraction from synaptosomal plasma membranes. The sphingosine and fatty acid composition allowed a
better interpretation of the MALDI-TOF analyses of the compounds because of the superposition of compounds of the same mass but different FAMEs and
sphingosine compositions. The nature of the different sphingosines was established through their mass obtained by GC/MS using the three different modes of
analysis.

Table V. Composition of standard glycoasparagines and reduced oligosaccharides

N-glycans
Man5GlcNAc
Man5GlcNAc2Asn
Man6GlcNAc2Asn
Man7GlcNAc2Asn
Man8GlcNAc2Asn
Man6GlcNAc3Asn
Fuc-di-antennary
Fuc-tri-antennary
Glycoprotein*a

Fuc
0.00
0.00
0.00
0.00
0.00
0.00
1.00
1.00
0.000

Reduced O-glycans
Rp N-4**

Fuc
1.00

Rp N-8*

0.00

Gal
0.00
0.00
0.00
0.00
0.00
0.00
2.01
3.00
2.996
(2.743)
Gal
1.99
(1.73)
2.99
(2.74)

Man
4.98
5.02
6.03
7.03
8.03
6.02
3.00
3.01
3.003

Glc
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.000

GlcNAc
1.00
1.01
1.01
1.01
1.01
2.02
3.02
4.02
2.996

GalNAc
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.994

NeuAc
0.00
0.00
0.00
0.00
0.00
0.00
2.02
3.01
3.003

Man
0.00

GlcNAc
0.10

GalNAc
1.00

NeuAc
0.00

GalNAc-OH
1.00

0.02

0.05

0.12

2.01

1.00

GN1
0.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.000

The determination was performed starting from 1 g of each compound submitted to methanolysis followed by acylation with HFBAA. Note that the
determination of the molar ration relative to that of the first GlcNAc residue allows differentiating without ambiguity the different oligomannosidic
glycoasparagines with a precision less than 1%. The same is true for complex type N-glycans and hybrid type N-glycans. The reduced O-glycans were isolated
from the mucins of the eggs from Rana palustris (Maes et al., unpublished observations).
aThis glycoprotein contained the following glycans:
NeuAc-Gal-GlcNAc-Man
Man-GlcNAc-GlcNAc-Asn-Prot
NeuAc-Gal-GlcNAc-Man

NeuAc-Gal-GalNAc-Ser-Prot

In compounds labeled * and **, a defect of the response of Gal was observed when the data were calculated based on the RMRmp (values into brackets). This
defect was no more observed when the area of the Gal peaks was obtained taking into accounts all Gal peaks and applying the RMR (see Table II). This was due
to the abnormal increase of the first Gal peak (Rt = 20.04) relative to the others in O-glycans.

The method was applied to a series of reduced oligosaccharides

with defined structures liberated by the reductive -elimination
procedure. The molar ratios obtained for all these compounds
approached the theoretical value at less than 1%, and sometimes
less than 0.1% (Table V). Surprisingly, in analysis of O-glycans
(reduced or not) in which the Gal residue is bound to GalNAc or
GalNAc-OH, the first furanic isomer of Gal (Rt = 20.04) showed
a significant relative increased compared to the other isomers, its
area being approximately the double of that obtained for standard
of galactose or for complex type N-glycans. This suggested that
during the cleavage of this particular glycosidic bond, the
formation of the O-methyl glycoside of this furanic form of Gal
is favored, a point that remains to be explained.

Analysis of the possible contaminants issued from glycoprotein

isolation procedures. Since the methods of glycoprotein purification
could introduce intrinsic contaminants, a special care was taken
to this point. Consequently, deoxycholate, sodium dodecyl
sulfate, Triton X-100, glycine, and Tris were submitted to the
complete procedure of methanolysis/HFBAA and analyzed by
GLC. A single peak corresponding to deoxycholate was eluted at
the end of the temperature gradient. The peaks of the Tris and
glycine derivatives were eluted close to the injection peak and
were in large part lost during the evaporation of the acylating
mixture because of their volatility. Triton X-100 is not significantly
degraded under the methanolysis conditions and gave three small
peaks without interferences with the monosaccharide derivatives.
261

J.-P.Zanetta, P.Timmerman and Y.Leroy

Fig. 3. Chromatogram of the monosaccharide composition of delipized synaptosomal plasma membranes isolated from adult rat brain. Due to the small amount of
GalNAc, it was suggested that O-glycans were minor glycans in these membranes. Note the presence of the peaks corresponding to the GlcNAc residue involved
in the N-glycosidic bond (GN1). No traces of glucuronic acid were found in this material, suggesting that the HNK-1 (glucuronic acid 3-sulfate) epitope is not
present on these purified plasma membranes. Although the membrane fraction was submitted to a drastic delipidation procedure, FAMEs were still detected in
high amount, but interestingly, the FAME composition was not the same as that of the lipids extract from the same fraction (Figure 1a). This suggested that an
important quantity of proteins or glycoproteins of these membranes are acylated by fatty acids. Note that after several injections, the separation between the Glc
and Gal isomers is almost complete.

In contrast, SDS gave peaks corresponding to compounds with

10, 12, 14, and 16 carbon atoms, the major peak of the C:12
component interfering with the isomer of mannose. Although
the relative proportions of the two Man isomers could not be
determined, the molar ratio of Man could be determined based on
the -Man area (93.05%). Consequently, these compounds used
for the purification of membrane-bound glycoproteins did not
interfere significantly with the analysis.
The sensitivity of the method theoretically allowed the
analysis of very small amounts of glycoproteins isolated by
electrophoresis. Therefore, it was of interest to examine if
polyacrylamide could interfere with the monosaccharide
determination. For this, a small piece of the polyacrylamide gel
(made in Tris-glycine buffer containing 0.1% SDS) was submitted to methanolysis followed by acylation. The complete
mixture gave only the peaks corresponding to SDS. In order to
test the possibility of performing a carbohydrate composition
directly on acrylamide gels, two glycoproteins (Thy-1 and CD24
isolated as concanavalin A-binding glycoproteins in young rat
cerebellum) were isolated by SDSPAGE. The gel was stained
with Coomassie Brilliant Blue R (solvents were of analytical
grade for this staining), the stained bands were dissected with a
lancet and transferred to the methanolysis vessel, and the gels
were dehydrated using repetitive washings in distilled methanol
(six times during 10 min). The gels were submitted to methanolysis
and after discarding the gels, the samples were evaporated under
nitrogen then analyzed after acylation. Although the molar ratios
of the different monosaccharides were identical to the values
obtained for the same glycoproteins recovered from the gel by
electroelution, the yield of the procedure was at least 10 times less
262

than expected. This was due to the absence of penetration of the

methanolysis reagent into the shrunken polyacrylamide gel (CBB
still remained at the center of the gel after 20 h of methanolysis).
Therefore, although the molar composition of the monosaccharides
could be obtained directly on a polyacrylamide gel, the poor
efficiency of the methanolysis procedure did not allow the
determination of the carbohydrate content of a glycoprotein.
This difficulty could be easily circumvented by blotting the
glycoproteins on PDVF membranes. After extensive washing with
water, the membrane was stained with Ponceau red, and then
washed again with water. The stained band was cut, and repetitively
washed with anhydrous methanol in the methanolysis vessel. After
methanolysis, the filter was discarded (after washing with methanol)
and the evaporated sample was acylated. In these conditions, the
methanolysis procedure allows the quantitative liberation of Omethyl glycosides. No interference occurred if the PDVF membranes were repetitively washed in methanol. The technique could
not be extended to nitrocellulose filters because of the complete
solubilization of nitrocellulose in methanol and in acetonitrile.
Because the carbohydrate analysis could be performed on
crude glycoprotein preparation or tissue extracts, we also
examined the possible contaminations induced by different
carbohydrate containing compounds. Consequently, RNA, DNA,
and a whole mixture of proteoglycans isolated from adult rat brain
in the absence of detergent (Normand et al., 1988), were
submitted to methanolysis in standard conditions followed by
derivatization with HFBAA. Only ribose isomers interfered with
the determination of fucose (Table I, Figure 1), but the presence
of ribose in a sample could be easily detected by the inversion of
the proportions of the two fuco-pyranose peaks. The small amount

HFB derivatives of O-methyl glycosides

of derivatives of uronic acids liberated from proteoglycans during

methanolysis (Table I) did not interfere with the separation of
other constituents (Table I). In contrast, a significant amount of
these compounds was liberated from branched glycans, in such a
way that the presence of specific peaks was indicative of the
presence of uronic acids in the glycans (Figure 1b). DNA
methanolysis products resulted in a black suspension upon
acylation, but again, none of the peaks interfered with the
determination of the classical glycoprotein monosaccharides.
Sensitivity of the technique and nanoanalysis
The method allowed the quantitative determination of the
carbohydrate composition of glycoproteins and glycolipids at the
level of the sensitivity of the FID detection, i.e., at 2550 pmol of
each monosaccharide injected onto the GLC column, for optimal
quantitations. In principle, since the totality of the sample can be
applied to the GLC column, analyses could performed on samples
containing such amounts of material. However, at this level,
dramatic cautions had to be taken with the purity of the reagents
and with the cleaning of the reaction vials, even with new vessels.
In our conditions, new vials contained a significant amount of
impurities that formed volatile derivatives eluted in the area of
FAME, whereas xylose and glucose were detected systematically.
Special sequentially redistilled reagents, absence of dust in the

laboratory, and special cleaning of the vessels are needed to

perform quantitative determinations starting from picomoles of
each glycoprotein or glycolipid monosaccharide.
Coupling with mass spectrometry
Although the reproducibility of the GC separation allowed to
assign the nature of the compound according to the retention
times of the isomers (all isomers of Ara, Rha, Rib, Xyl, Fuc, Gal,
Man, Glc, GlcNAc, GalNAc, KDN, Neu5Ac are separated, and
separated from glucitol, mannitol, inositol, N-acetyl-glucosaminitol,
N-acetyl-glucosaminitol, and uronic acids; from the fatty acid
methyl esters larger than C14:0; from sphingosine bases; and
from major contaminants found during glycoprotein and glycolipid
isolation), the absolute identification of each compounds in
samples with an unknown composition needs mass spectrometry.
The HFB derivatives of the O-methyl glycosides of monosaccharides presented a relatively high mass (978 for hexoses, 977
for hexosamines, 1275 for sialic acid). Chemical ionization (NH3
gas) MS analysis of the compounds performed with detection of
positive ions, giving the pseudo-molecular ion [M + NH4]+, was
a very secure identification of the families of compounds
(Table VI), provided that the mass limit of the apparatus was
2000 amu. Unfortunately, most of the routine GC/MS systems are
out of this range.

Table VI. Reporter fragments of monosaccharides in the EI/MS and CI/MS modes
EI (70 eV)
calc. molec. ion

Derivatives of
HFB
Pentoses
Deoxy-hexoses
Hexoses
Hexosamines
Sialic acid
KDN
Uronic acids
meso-inositol
mannitol
GlcNAc-OH
GalNac-OH
GlcN*
Asp
Lys
Sphingosines

C18:1
C20:1
C22:1
C24:1
C18:0
C20:0s
C22:0s

752
766
978
977
1275
1276
810
1356
1358
1357
1357
963
357
552
887
915
943
971
889
705
733

Specific ions
observed
69119169
479265325
279492
551519337277
276472488702
542602789
575515355
323537597
715
503453
488
488
476348290
236299357
520280
459
487
515
543
n.d.
n.d.
n.d.

Positive CI(NH3) calc. pseudo

molec. ion (M+NH4)+ and observed ions

Negative CI(NH3)
observed ions

770
784
996
995
1293
1294
828
1374
1376
1375
1375
981
375
570
905
933
961
989
n.d.
n.d.
n.d.

752
766
978958
977
n.d.
n.d.
810790
n.d.
n.d.
n.d.
n.d.
963943
357
552
887
915
n.d.
n.d.
889
685
713

770
784
996
995
1293
1294
828
1374
1376
1375
1375
981963
375
570
905691*
933719*

For electron impact (EI/MS), the indicated ions correspond to those that allowed the identification of families of compounds or individual compounds. The
scheme of ionization involved primarily a loss of one or two heptafluorobutyric acid(s) (mass decay = 214), and frequently 31 (O-CH3). For uronic acid, an
additional loss of the carboxyl group in the C(6) position occurred. When several ions are reported, the higher mass entity was the more discriminative, but
because it was present in relatively low proportion, it did not allow the detection of low amounts. Sphingosines gave preferentially ions corresponding to
[M 2 214] (M two heptafluorobutyric acids). Furanic forms of oses and uronic acids can be specifically identified by a reporter ion at 525, whereas the
furanic forms of hexosamines are characterized by the ion at 524.
In the CI/MS positive mode, the indicated mass corresponded to [M +18]+. * Intense ions were observed corresponding to [M + 18 - 214] for sphingosines. A
fragment at M = 867 allows the identification of the derivatives of NeuAc using the Finnigan Automass II mass spectrometer with the mass limit at 1000 amu.
In the CI negative mode, the ions corresponded generally to [M]
. A loss of mass of 20 was also observed for some compounds corresponding to the loss of
one HF molecule. An ion at M = 809 allowed the characterization of the NeuAc derivative on the Finnigan Automass II device. Sphingosines gave [M]
or the
same with a loss of 20. C20:0s and C22:0s were di-hydro-sphingosines found in a tri-sialo-gangliosides isolated from synaptosomal plasma membranes
(unpublished observations) in which the hydroxyl group is replaced by a hydrogen atom. n.d., Not determined.

263

J.-P.Zanetta, P.Timmerman and Y.Leroy

Fig. 4. Reporter ion analysis in the EI mode of a mixture of pentoses, deoxy-hexoses, hexoses, uronic acids, N-acetyl-hexosamines, and sialic acid submitted to
methanolysis then acylation with HFBAA. Note that specific ions allowed identifying the different families of compounds.

In contrast with the TMS derivatives, HFB derivatives of

O-methyl glycosides of sugars gave secure information under the
electron impact mode, allowing the identification of fragments
specific for families of compounds with a routine apparatus with
a mass limit of 1000 amu. Because of the presence of
characteristic peaks due to the HFB part (M = 69 (CF3+), 119
(CF2-CF3+), and 169 (CF2-CF2-CF3+)), it could be ascertained
that the compound was an heptafluorobutyrate derivative and
contained hydroxyl or amino groups. Furthermore, using the
EI/MS mode, specific ions were reproducibly obtained for each
family of compounds (Table VI), independent on the quantity of
material. At the present status, the nature of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, sialic acid and
KDN, aspartic acid, the internal standard lysine, fatty acid methyl
esters, and sphingosines can be identified using a selective
research of characteristic ions by chromatogram reconstitution.
(Figures 2, 4). The presence of specific ions for the furanic forms
of the O-methyl glycosides of Fuc, Gal, GalNAc, and uronic acids
(legend of Table VI) reinforced the identification of the different
compounds.
Because of the presence of a large number of fluorine atoms
in each derivative, we tested the chemical ionization mode (NH3
gas) with a detection of negative ions using the Automass II device
(mass limit 1000 amu). The different compounds can be detected by
intense ions corresponding to [M]
ion and/or [M 20]
ion, the
latter issued from an association resonance capture of an electron
264

with the elimination of a HF molecule. Although this detection

did not allow obtaining signals for FAME, it appeared to be an
extremely sensitive detection technique (more than 20 times that
of the positive ion detection), not only for the HFB derivatives of
O-methyl glycosides, but also for those of sphingosines and of
hydroxylated fatty acid methyl esters.
Conclusions and perspectives
The method described here for the determination of the monosaccharide composition of glycoprotein and glycolipids provides
important advantages compared to those previously described.
The major features are simplicity of the technique, the stability of
the derivatives, and the quasi-absence of interferences by
classical contaminants. The quantitation of the hexosamine and
sialic acid content is by far more reliable than that measured by
the classical TMS method, since there is no requirement for
re-acetylation. Although the molar response of the HFB derivatives
is 50% lower than that of the TMS derivatives, the stability of the
HFB derivatives allows one to inject (if necessary), after
concentrating under a stream of nitrogen, the whole sample on the
column. The quantitation available for the GlcNAc residue
involved in the N-glycosidic bond as a separated compound
provides an essential information on the structure of N-glycans.
As demonstrated elsewhere (Maes et al., 1999), the N-glycosidic
bond is cleaved with 96% yield under our methanolysis
conditions. Several compounds are produced corresponding

HFB derivatives of O-methyl glycosides

essentially to the and isomers of glucosamine that are clearly

separated from the O-methyl glycosides of glucosamine, which are
derived from O-linked GlcNAc. Although these compounds can be
detected on chromatograms of TMS derivatives, their quantitation
remains impossible because of uncontrolled degradation within
the GC injector and interference with -Glc; Maes et al., 1999).
Because of the absence of large interferences stemming from
buffers and polyacrylamide, the method allows the quantitative
determination of the glycoprotein monosaccharide composition
with high sensitivity. Indeed, less than 1 g of glycoprotein
purified from acrylamide or on PDVF membranes is required. As
observed recently in our laboratory, the exact carbohydrate
composition can be obtained directly from a glycoprotein
dissolved in phosphate-buffered saline without interference. For
glycoproteins and glycolipids, not only is the carbohydrate
composition accessible but one can also obtain that of the fatty
acids methyl esters and that of sphingosines. Specific ions in the
EI/MS mode and the molecular mass in the CI/MS modes can be
reproducibly obtained, thus nailing down the nature of the
monosaccharides, and that of other constituents of glycoconjugates such as FAMEs and sphingosine bases. Works in the field
of mass spectrometry are now in progress to identify specific ions
allowing the identification of each isomer of the heptafluorobutyrate derivatives of all O-methyl glycosides. Because of the
reproducibility of the spectra, independent on the quantity of
material loaded of the GC/MS device, it is expected that computer
storage of the data of these spectra can allow a clear and rapid
identification of the different compounds using routine GC/MS
systems.

Materials and methods

Chemicals
Standard monosaccharides and sodium deoxycholate were from
Sigma Chemical Co. (St. Louis, MO); heptafluorobutyric and
pentafluoropropionic anhydrides were from Fluka (Buchs,
Switzerland). SE-30 liquid phase and the Gas Chrom Q solid
phase were purchased from Applied Science Inc. (State College,
PA). The capillary columns (25 m 25QC3/BP1, 0.5 m film
phase) were from SGE France SARL (Villeneuve St. Georges,
France). Purified calcinated calcium chloride was from Prolabo
(Paris, France) and HPLC grade acetonitrile, TRIS, and acrylamide
were from SDS (Peypin, France). Gangliosides GM1 was
purified from the rat cerebellum (Zanetta et al., 1980), and
ganglioside G5b was a generous gift of Prof. G. Tettamanti.
Reduced oligosaccharides were kindly provided by Dr. E. Maes
(Maes et al., unpublished observations). Fatty acid methyl esters
were prepared from the synaptosomal plasma membranes of
adult rat brain (Breckenridge et al., 1972). For use as an internal
standard, lysine hydrochloride (Sigma) was recrystallized from a
1 mM HCl solution in water.
Methanolysis
Samples containing the internal standard (0.22 g lysine) were
lyophilized in conical heavy walled Pyrex tubes (2.0 ml) with
Teflon-lined screw caps. 0.250.5 ml of the methanolysis reagent
was added, and the closed vessels were left for 20 h at 80
C. The
methanolysis reagent was obtained by dissolving anhydrous
gaseous HCl (up to 0.5 M) at -50
C in anhydrous methanol
previously redistilled on magnesium turnings (Zanetta et al.,

1972). Gaseous HCl was prepared by the dropwise addition of

concentrated sulfuric acid on crystallized sodium chloride.
Acylation
After methanolysis, samples were evaporated to dryness under a
light stream of nitrogen in a ventilated hood, followed by the
addition of 200 l acetonitrile and 25 l of heptafluorobutyric
anhydride (HFBAA) with a pipette with plastic tips. The closed
vessels were heated for 30 min at 100
C (or better for 5 min at
150
C in order to have a reflux reaction that derivatizes the traces
of compounds on the vessel wall) in a sand bath. After cooling at
room temperature, the samples can be stored for months at room
temperature in the closed vessel without need of reacylation.
When analysis had to be performed, the samples were evaporated
in a light stream of nitrogen in a ventilated hood in order to
eliminate the excess of reagent and the heptafluorobutyric acid
(HFBA) formed during the acylation, then taken up in the
appropriate volume of acetonitrile; this acetonitrile was stored in
a closed vessel in the presence of calcinated calcium chloride in
order to eliminate traces of water. An aliquot of the acetonitrile
solution of the heptafluorobutyrate derivatives (HFB) was
introduced in the Ross injector of the GC apparatus. Although we
did not observed changes in the RMR of the different derivatives
after storage for 2 days at room temperature of the samples in
anhydrous acetonitrile, we do not recommend storing the
derivatives in acetonitrile, but in the acylation mixture.
Gas chromatography
For preliminary studies of the volatility and stability of the HFBF
derivatives, analyses were performed on a 3 m long column packed
with 3% SE-30 (Applied Science Inc., State College, PA) on Gas
chrom Q (Applied Science Inc.). Injector and detector temperature
was 260
C; and the temperature program was 2
C/min from 100
to 240
C at a helium flow rate of 20 ml/min. For analytical
purposes, analyses were performed on a Shimadzu GC-14A gas
chromatograph equipped with a Ross injector and a 25 m long
capillary column (25QC3/BP1; 0.5 m film phase; SGE France
SARL; Villeneuve St. Georges (France). Injector and flame
ionization detector temperatures were 260
C and the temperature
program was 1.2
C/min between 100 and 140
C, followed by
4
C/min from 140
C to 240
C then maintaining this temperature
for 10 min. The carrier gas (helium) pressure was 0.8 bar. The
second part of the temperature program is only partially involved in
the separation of monosaccharide derivatives (except KDN and
sialic acid), but can reveal the presence of additional compounds,
especially fatty acids or sphingosines present in glycolipids and also
in glycoproteins (GPI anchors, myristylation, etc.).
Mass spectrometry
For GC/MS analysis, the GLC separation was performed on a
Carlo Erba GC 8000 gas chromatograph equipped with a 25 m
0.32 mm CP-Sil5 CB Low bleed/MS capillary column, 0.25 m
film phase (Chrompack France, Les Ullis, France). The temperature
of the Ross injector was 260
C and the samples were analyzed
using the following temperature program: 90
C for 3 min then
5
C/min until 240
C. The column was coupled to a Finnigan
Automass II mass spectrometer or, for mass larger than 1000, to
a Riber 1010H mass spectrometer (mass detection limit 2000).
The analyses were performed either in the electron impact mode
(ionization energy 70 eV; source temperature 150
C) or in the
chemical ionization mode in the presence of ammonia (ionization
energy 150 eV, source temperature of 100
C). The detection was
265

J.-P.Zanetta, P.Timmerman and Y.Leroy

performed for positive ions or for negative ions, in separated

experiments, the latter allowing the quite specific detection of
heptafluorobutyrate derivatives with a higher sensitivity.
Acknowledgments
We thank Drs. J.-C.Michalski, G.Strecker, J.Lemoine, E.Maes,
O.Kol, and Mrs. A.Copin for providing oligosaccharides and
glycoproteins of known monosaccharide compositions and Mrs.
C.Alonso for help in iconography.
Abbreviations
Ara, arabinose; Rha, rhamnose; Xyl, xylose; Fuc, fucose; Rib,
ribose; Gal, galactose; Man, mannose; GlcNAc, glucosamine; GalNac, N-acetyl-galactosamine; ManNAc, N-acetylmannosamine; GalNAc-OH, reduced GalNAc; GlcNAc-OH,
reduced GlcNAc; GlcA, glucuronic acid; GalA, galacturonic acid;
NeuAc, N-acetyl neuraminic acid; KDN, 3-deoxy-D-glycero-Dgalacto-nonulosonic acid; Meso, meso-inositol; Manni, mannitol;
Asp, aspartic acid; Lys, lysine. HFB, heptafluorobutyrate; HFBAA,
heptafluorobutyric anhydride; TFA, trifluoroacetate; EI, electron
impact; CI, chemical ionization; MS, mass spectrometry.

266

References
Breckenridge,W.C., Gombos,G. and Morgan,I.G. (1972) The lipid composition of
adult rat brain synaptosomal plasma membranes. Biochim. Biophys. Acta,
266, 695707.
Chaplin,M.F. (1994) Monosaccharides. In Chaplin,M.F. and Kennedy,J.F. (eds.),
Carbohydrate Analysis. A Practical Approach. Oxford Universtity Press,
Oxford, pp. 141.
Chiba,A., Matsumura,K., Yamada,H., Inazu,T., Shimizu,T., Kunusoki,S.,
Kanazawa,I., Kobata,A. and Endo,T. (1997) Structures of sialylated O-linked
oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a
novel O-mannosyl-type oligosaccharide in the binding of alpha dystroglycan
with laminin. J. Biol. Chem., 272, 21562162.
Maes,A., Strecker,G., Timmerman,P., Leroy,Y. and Zanetta,J.-P. (1999) Quantitative
cleavage of the N-glycosidic bond in the normal conditions of methanolysis
used for the analysis of glycoprotein monosaccharides. Anal. Biochem., in press.
Normand,G., Kuchler,S., Meyer,A., Vincendon,G. and Zanetta,J.-P. (1988)
Isolation and immunohistochemical localization of a chondroitin sulfate
proteoglycan from adult rat brain. J. Neurochem., 51, 665676.
Zanetta,J.-P. and Vincendon,G. (1973) Gas-liquid chromatography of the
N(O)-heptafluorobutyrates of the isoamyl esters of amino acids. I. Separation
and quantitative determination of the constituent amino acids of proteins.
J. Chromatogr., 76, 9199.
Zanetta,J.-P., Breckenridge,W.C. and Vincendon,G. (1972) Analysis of monosaccharides by gas-liquid chromatography of the O-methyl glycosides as their
trifluoroacetate derivatives Application to glycoproteins and glycolipids. J.
Chromatogr., 69, 291304.
Zanetta,J.-P., Vitiello,F. and Vincendon,G. (1980) Gangliosides from rat cerebellum:
demonstration of a considerable heterogeneity using a new solvent for
thin-layer chromatography. Lipids, 15, 10551061.