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Clinical Chemistry 44: 2422-2428, 1998;
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(Clinical Chemistry. 1998;44:2422-2428.)
© 1998 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

André Klein1,a, Alexandra Lebreton1, Jérome Lemoine2, Jean-Marc Périni1, Philippe Roussel1 and Jean-Claude Michalski2

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The extreme diversity of oligosaccharides that can be found in urine renders their analysis and screening extremely difficult. Oligosaccharides such as lactose, sialyllactose, or glucose tetrasaccharide (Glc4),1 or oligosaccharides bearing A, B, or O blood group determinants are commonly found in nonpathological urine samples (1)(2). Increased oligosacchariduria is seen in the urine of lactating or pregnant women, breast milk-fed neonates, or preterm infants fed with milk fortified with Glc polymers (1)(3)(4). Abnormal excretion of oligosaccharides is found in glycoprotein degradation disorders and in some glycogen storage diseases (5)(6). In these lysosomal diseases, analysis of urinary oligosaccharides is used as a diagnostic aid.

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.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
ANTS was obtained from Molecular Probes, Inc.; amyloglucosidase from Aspergillus niger and {alpha}-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 {alpha}-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.).


   Results
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 Materials and Methods
 Results
Discussion
 References
 
A typical electrophoresis of ANTS-derivatized maltooligosaccharides after purification on an SPE column is shown in Fig. 1 . 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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  		Figure 1. Oligosaccharide polyacrylamide gel electrophoresis of ANTS-derivatized oligosaccharides purified over a PGC column.

Lane 1, oligosaccharide ladder (mixture of lactose, maltotriose, and maltooligosaccharides (G4–G12); lane 2, maltotetraose; lanes 3–5, fractions I, II, and III obtained by purification of the coupled maltooligosaccharides (G4–G12) on a PGC column.

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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  		Figure 2. MALDI-TOF mass spectrum of maltooligosaccharides.

Approximately 50 pmol of the oligosaccharide mixture was loaded on the target. The indicated m/z values correspond to pseudomolecular ions corresponding to the loss of one H, with chemical compositions as given in Table 1Up .


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  		Table 1. Monosaccharide composition of the ANTS-derivatized urinary oligosaccharides deduced from the m/z values of the pseudomolecular ions observed in the different samples.

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 1Up . 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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  		Figure 3. MALDI-TOF mass spectra of urinary oligosaccharides.

(A) nonpathological urine; (B) nonpathological urine of a patient receiving cornstarch as a food supplement; (C–G), pathological urines (C, Pompe disease; D, {alpha}-mannosidosis; E, ß-mannosidosis; F, galacto-sialidosis; G, GM1 gangliosidosis). The indicated m/z values correspond to pseudomolecular ions resulting from the loss of one H, with chemical compositions as given in Table 1Up .

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. 3AUp .

Food supplementation with cornstarch causes the urinary excretion of Glc polymers. In Fig. 3BUp , 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 {alpha}-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. 3CUp ) 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.

{alpha}-Mannosidosis.
The pseudomolecular ions observed in the mass spectrum of the ANTS-derivatized oligosaccharides of a patient suffering from {alpha}-mannosidosis (Fig. 3DUp ) 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 Man{alpha}1->3Manß1->4GlcNAc (6)(17).

ß-Mannosidosis.
The spectrum of the ANTS-oligosaccharides of a patient suffering from ß-mannosidosis (Fig. 3EUp ) was characterized by a major signal at m/z 749, which could be identified as Manß1->4GlcNAc (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. 3FUp ) 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 1Up . The major pseudomolecular ions and the corresponding oligosaccharide structures are given in Table 2 (6).


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  		Table 2. Structure and values of the major pseudomolecular ions accumulating in urine of the patients with GM1 gangliosidosis (A) and galacto-sialidosis (B).

GM1 gangliosidosis.
The oligosaccharides present in the urine of the GM1 gangliosidosis patient (Fig. 3GUp ) 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 1Up . The major pseudomolecular ions and the corresponding structures in GM1 gangliosidosis are given in Table 2Up .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A rapid and simple procedure of SPE on a PGC column described by Packer et al. (15), which allowed easy clean-up of the sample after the coupling reaction, was used to separate the coupled oligosaccharides from urinary noncarbohydrate components and from coupling reagent. We took advantage of the negative charges of the three sulfonic groups of ANTS coupled to the oligosaccharides, which increased retention to the PGC. The ANTS-derivatized neutral or sialylated oligosaccharides were eluted only by the third eluent, which contained TFA.

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 1Up ). 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 2Up ) 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 {alpha}-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.


   Footnotes
 
1 Nonstandard abbreviations: Glc, glucose; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; SPE, solid phase extraction; PGC, porous graphite carbon; TFA, trifluoroacetic acid; amu, atomic mass unit; Hex, hexose; HexNAc, N-acetylhexosamine; Man, mannose; and GlcNAc, N-acetylglucosamine.


   References
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 Abstract
 Introduction
 Materials and Methods
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 Discussion
 References
 

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