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Molecular Diagnostics and Genetics |
1
Centre Hospitalier Régional Universitaire de Lille, Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Calmette, Bd du Professeur Jules Leclercq, 59047 Lille Cedex, France.
2
Laboratoire de Chimie Biologique, CNRS Unité Mixte
111, Université des Sciences et Technologies de Lille, 59655
Villeneuve d'Asq Cedex, France.
a Author for correspondence. Fax 33 3 20 53 85 62; e-mail klein{at}lille.inserm.fr.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most current assays for oligosaccharides use thin-layer chromatography (7) and HPLC (7)(8). HPLC is more sensitive and provides higher resolution than thin-layer chromatography; however, carbohydrates are revealed on thin-layer chromatography by specific staining procedures, whereas HPLC often uses nonspecific detection unless postcolumn derivatization is used (8). Recently, fluorophore-assisted carbohydrate electrophoresis was developed for the identification of lysosomal storage diseases: oligosaccharides are labeled with a fluorescent tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), by reductive amination, and then separated by polyacrylamide gel electrophoresis (9). The electrophoretic data are interpreted by "oligosaccharide profiling" and permit the initial screening of the lysosomal storage diseases (10). Precise identification of urinary oligosaccharides can be achieved after purification of the oligosaccharides and compositional analysis and physico-chemical techniques such as nuclear magnetic resonance or mass spectrometry [for reviews of these methods, see Refs. (1)(6)]. These approaches, however, are long and tedious. Another approach is the use of exoglycosidases to remove the terminal nonreducing monosaccharide; the interpretation of the modified electrophoretic or HPLC pattern gives a structural indication of the nature of the oligosaccharide; this method, therefore, is very useful. Nevertheless, in some cases the interpretation of profiles is difficult because of unsuccessful or incomplete enzymatic digestions, the diversity of the excreted structures, the purity of the exoglycosidases, and the necessary use of a large number of different enzymes (11)(12).
We here propose the use of a sensitive procedure, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ANTS-derivatized oligosaccharides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amylase from Bacillus
amyloliquefasciens were from Boehringer Mannheim. All other
chemicals were of reagent grade and were purchased from commercial
sources.
urine specimens
Urine samples were obtained from patients with various lysosomal
diseases, from neonates, and from healthy control persons. Urines were
stored frozen until used. In cases of galacto-sialidosis and GM1
gangliosidosis, the oligosaccharides had been characterized and
quantified previously (6)(13).
derivatization of oligosaccharides with ants
Reductive amination of oligosaccharides with ANTS was performed as
described by Jackson (14). Two 100-µL urine aliquots were
dried in 1.5-mL microcentrifuge tubes in a centrifugal vacuum
evaporator. Each dried residue was resuspended in 5 µL of 0.15 mol/L
ANTS in a solution of 150 mL/L acetic acid–850 mL/L water, and 5 µL
of sodium cyanoborohydride (0.1 mol/L) in dimethyl sulfoxide; the
samples were labeled overnight (16 h) at 37 °C. The reaction
mixtures were dried, resuspended in 1 mL of water, and pooled. The
pooled mixtures were purified by solid phase extraction (SPE) on a 1-mL
column containing porous graphite carbon (PGC; 300 mg of graphite;
Hypercarb®; Shandon Scientific), using a procedure
analogous to the one described by Packer et al. (15).
Briefly, the column was washed before use with 5 mL of 500 mL/L
acetonitrile–500 mL/L water containing 1 g/L trifluoroacetic acid
(TFA), followed by 10 mL of water. The pooled mixtures were loaded onto
the adsorbent at a flow rate of 1 mL/min; salts and excess reagents
were washed off with 5 mL of water (fraction I) and 5 mL of 150 mL/L
acetonitrile–850 mL/L water (fraction II); and labeled
oligosaccharides were eluted with a third solvent, 250 mL/L
acetonitrile–750 mL/L water containing 1 g/L TFA (fraction III).
Electrophoresis of the different fractions was performed to control the
elution.
electrophoresis of ants-derivatized oligosaccharides
The method used to separate ANTS-derivatized oligosaccharides used
the Tris-glycine discontinuous buffer system of Laemmli
(16), except that the sodium dodecyl sulfate and the
2-mercaptoethanol were omitted from all buffers. Isocratic running gels
containing 350 g/L acrylamide and 9.3 g/L
N,N'-methylenebisacrylamide were polymerized with 25 µL of
an aqueous solution of 100 g/L ammonium persulfate and 10 µL of
N,N',N',N'-tetramethylene diamine. The electrophoretic
conditions were as described by Jackson (13). A digitized
image of the gel was recorded by an imaging system based on a cooled,
charge-coupled device camera and a standard ultraviolet light box.
enzyme digestions
A sample of urine (1 mL) was purified using an SPE PGC column.
Fraction II, eluted with 250 mL/L acetonitrile–750 mL/L water,
contained the urinary oligosaccharides and was evaporated and
resuspended in 1 mL of water. To 5 µL of fraction II was added 5 µL
of 0.1 mol/L ammonium acetate, pH 5.5, and 1.35 U of -amylase from
B. amyloliquefasciens.
To another 5 µL of fraction II was added 5 µL of 0.1 mol/L phosphate-citrate buffer, pH 5, and 0.5 mg of amyloglucosidase from A. niger. Digestions were performed for 2 h at 37 °C. The reactions were stopped with 1 mL of cold ethanol, dried on a centrifugal vacuum evaporator, labeled with ANTS, and submitted to polyacrylamide gel electrophoresis.
maldi-tof ms
Mass spectrometric analysis was carried out on a Vision 2000
(Finnigan) instrument operating in negative linear mode at an
accelerating voltage of 20 kV. The matrix solution was prepared by
dissolving 10 mg of 3-aminoquinoline in 1 mL of 2 mmol/L ammonium
acetate in 700 mL/L methanol–300 mL/L water. Samples were dissolved in
250 µL of water. A volume of 0.5 µL of the sample was mixed
directly on the target with 1.5 µL of the matrix solution and then
dried under vacuum. Approximately 15–20 shots from the pulsed laser
(pulse time, 3 ns) were accumulated to get the final spectrum. External
calibration was done with angiotensin I (Mr
1296.7; Sigma Chemical Co.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The ANTS-oligosaccharides were found in fraction III, eluted
with 250 mL/L acetonitrile–750 mL/L water–1.0 g/L TFA; fractions I
and II, which contained salts and excess reagents, did not contain
oligosaccharides.
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The MALDI-TOF MS spectrum of ANTS-maltooligosaccharides recorded in the
negative mode is shown in Fig. 2
. The ions observed correspond to molecular ions that have lost
one proton, [M-H]-, and are therefore called
pseudomolecular ions. All of the pseudomolecular ions observed were
separated by a 162-atomic mass unit (amu) difference, which
corresponded to an hexose mass minus a molecule of water for the
linkage. Chemical compositions deduced from the different
pseudomolecular ions [M-H]- are given in Table 1
.
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The spectra obtained in different nonpathological and pathological
cases (described below) are shown in Fig. 3
. The chemical compositions of the pseudomolecular ions are
given in Table 1
. The incremental residue masses for the commonly found
residues are 162 amu for a hexose (Hex), 146 amu for a deoxyhexose
(deoxyHex), 203 amu for an N-acetylhexosamine (HexNAc), and
291 amu for N-acetylneuraminic acid.
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nonpathological urines
Usually, nonpathological urine does not contain a substantial
amount of oligosaccharide. Nevertheless, various amounts of lactose,
sialyllactose, and a glucotetrasaccharide (Glc4) may
sometimes be observed, with the corresponding ions at m/z
708, 999, and 1032. An example of a nonpathological urine is given in
Fig. 3A
.
Food supplementation with cornstarch causes the urinary excretion of
Glc polymers. In Fig. 3B
, a typical ANTS-oligosaccharide profile of
urine from a subject on a cornstarch-supplemented diet is shown, with
the presence of pseudomolecular ions [M-H]- at
m/z 870, 1032, 1194, and 1518 corresponding to
oligosaccharides Glc3, Glc4, Glc5,
and Glc7, respectively. In the same urine, a
pseudomolecular ion at m/z 999, corresponding to
sialyllactose, is observed. Complete identification of the Glc polymers
as limit-dextrins was done enzymatically; amyloglucosidase destroyed
all of the Glc polymers, whereas -amylase did not affect the
profiles (data not shown).
oligosaccharidosis urines
Pompe disease.
The profile of the ANTS-derivatized
oligosaccharides from a patient with Pompe disease (Fig. 3C
) was
characterized by a major pseudomolecular ion at m/z 1032,
which corresponds to a Glc tetrasaccharide (Glc4). All
pseudomolecular ions of Glc polymers with a degree of polymerization up
to 11 were also observed.
-Mannosidosis.
The pseudomolecular ions observed in the
mass spectrum of the ANTS-derivatized oligosaccharides of a patient
suffering from -mannosidosis (Fig. 3D
) were at m/z 911,
1073, 1235, 1397, 1559, 1721, and 1883. Their interpretation gave a
chemical composition of HexnHexNAc1
(n = 2–8) for the different oligosaccharides and corresponded to
MannGlcNAc1. The major compound was the
trisaccharide at m/z 911, corresponding to
Man1
3Manß14GlcNAc (6)(17).
ß-Mannosidosis.
The spectrum of the ANTS-oligosaccharides of
a patient suffering from ß-mannosidosis (Fig. 3E
) was characterized
by a major signal at m/z 749, which could be identified as
Manß14GlcNAc (6). No chemical compositions could be
deduced from two minor ions present at m/z 1148 and 1503.
Galacto-sialidosis.
The spectrum of the ANTS-derivatized
oligosaccharides of a patient with galacto-sialidosis (Fig. 3F
) was
characterized by two major pseudomolecular ions at m/z 2385
and 1567; minor pseudomolecular ions were detected at m/z
1202, 1276, 1729, and 2020, and an intense pseudomolecular ion at
m/z 708 probably corresponds to lactose. The chemical
composition deduced from these ions is listed in Table 1
. The major
pseudomolecular ions and the corresponding oligosaccharide structures
are given in Table 2
(6).
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GM1 gangliosidosis.
The oligosaccharides present in the urine
of the GM1 gangliosidosis patient (Fig. 3G
) had a specific mass
spectrum, with major pseudomolecular ions at m/z 708, 854,
1276, 1803, and 2170, and less intense ions at 1219, 1438, and 1641.
The chemical compositions deduced from these ions are listed in Table 1
. The major pseudomolecular ions and the corresponding structures in
GM1 gangliosidosis are given in Table 2
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purified ANTS-derivatized oligosaccharides were analyzed by a
MALDI-TOF MS technique. The negatively charged sulfonic groups
permitted the detection of the oligosaccharides in the negative mode.
Because of the mild ionization process, each ANTS-derivatized
oligosaccharide produced only one ion, corresponding to
[M-H]-, and mixtures could be analyzed in one
experiment. The carbohydrate composition was determined from the value
of the pseudomolecular ion of each oligosaccharide (Table 1
). The
amount of derivatized oligosaccharides loaded on the target of the
MALDI-TOF mass spectrometer could be estimated from the previously
determined carbohydrate chemical composition of the same urines
(6)(13). The concentration of oligosaccharides
BI and BII were ~110–120 µmol/L and 60
µmol/L, respectively, and the mass spectral pattern revealed a
representative picture of the urinary oligosaccharide distribution; a
similar observation was made with GM1 gangliosidosis urines
(13). In contrast to techniques that use internal standards,
such as HPLC or gas chromatography–mass spectrometry, the patterns
obtained with this MALDI-TOF MS technique are more qualitative than
quantitative. The detection limit of the technique could be estimated
in the very low picomolar range: minor oligosaccharides
(AIII and AIV in Table 2
) present in the urine
at concentrations of ~5 µmol/L (13) could be identified
easily. This result is in accordance with previously described
detection limits (18)(19)(20).
Identifying the oligosaccharides present in each lysosomal storage disease on the basis of the values of the pseudomolecular ions and their intensity was easy: in Pompe disease, the presence of Glc polymers up to a degree of polymerization of 11 with a very intense peak corresponding to a Glc tetrasaccharide was characteristic and could be distinguished from urines of subjects receiving cornstarch as a food supplement. In this case, the oligosaccharides were in lower quantities and the most intense pseudomolecular ions were the Glc tri- and tetrasaccharides. The Glc penta- and hexasaccharides were present in very low concentrations, and higher-sized Glc polymers were not detected.
ANTS-derivatized oligosaccharides from -mannosidosis, GM1
gangliosidosis, and galacto-sialidosis urines have very specific
patterns of excreted oligosaccharides; nevertheless, the confirmation
of these diseases should be made by enzymatic or genetic analysis. It
should also be mentioned that other glycoprotein degradation disorders,
such as aspartylglucosaminuria or Schindler disease
(6)(21), are characterized by abnormal excretion
of glycopeptides and cannot be identified by this technique.
HPLC (8) or electrophoresis (10) provide only an oligosaccharide pattern, whereas the MALDI procedure described in the present work allows identification of the chemical composition of each peak observed. In the case of ambiguous pseudomolecular ions, structural identification of the compound can be achieved by sequential digestion of the ANTS-oligosaccharides by selectively chosen exoglycosidases (11).
This fast and sensitive technique should allow screening of numerous unclassified disorders characterized by urinary excretion of oligosaccharides, such as myoclonic encephalopathy (22), and may also be extended to other pathologies.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6Galß14GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis. Eur J Biochem 1991;198:521-526.
[ISI][Medline]
[Order article via Infotrieve]
-N-acetylgalactosaminidase deficiency. N Engl J Med 1989;320:1735-1740.
[ISI][Medline]
[Order article via Infotrieve]
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