Structural characterization of the N-linked oligosaccharides in bile salt-stimulated lipase originated from human breast milk

Yehia Mechref2, Peng Chen and Milos V. Novotny1

Department of Chemistry, Indiana University, Bloomington, IN 47405, USA

Received on January 6, 1998; revised on July 13, 1998; accepted on July 13, 1998

The detailed structures of N-glycans derived from bile salt-stimulated lipase (BSSL) found in human milk were determined by combining exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-glycan structures were conclusively determined in terms of complexity and degree of fucosylation. Ion-exchange chromatography with pulsed amperometric detection, together with mass-spectral analysis of the esterified N-glycans, indicated the presence of monosialylated structures. The molecular mass profile of esterified N-glycans present in BSSL further permitted the more detailed studies through collision-induced dissociation (CID) and sequential exoglycosidase cleavages. The N-glycan structures were elucidated to be complex/dibranched, fucosylated/complex/dibranched, monosialylated/complex/dibranched, and monosialylated/fucosylated/dibranched entities.

Key words: bile salt-stimulated lipase/MALDI/TOF-MS/exoglycosidases/N-glycans

Introduction

Bile salt-stimulated lipase (BSSL) is an enzyme with a broad substrate specificity toward mono-, di-, and triacylglycerols; cholesterol esters; fat-soluble vitamins; and lipoamides (Bernbäck et al., 1990; Hui et al., 1993; Chen et al., 1994). The enzyme represents the major lipolytic activity in the milk and is also important in the energy uptake (Hernell and Olivecrona, 1974). BSSL is a glycoprotein which amounts to approximately 0.5-1.0% of the milk protein (Hernell and Olivecrona, 1974; Bläckberg and Hernell, 1981). The biological roles of BSSL glycans are speculative at present, since glycosylation does not seem to effect the main catalytic activity of this enzyme (Hansson et al., 1993; Bläckberg et al., 1995).

The glycoprotein consists of 722 amino acid residues, with one potential N-glycosylation site at Asn-187 at the N-terminus (Nilsson et al., 1990; Baba et al., 1991). Toward the C-terminus, however, the residues with potential O-glycosylation sites abound (Nilsson et al., 1990; Baba et al., 1991). While the amino acid sequence of BSSL appears identical to the pancreatic bile salt-dependent lipase (also known as pancreatic cholesterol esterase; Nilsson et al., 1990), the two enzymes differ in their molecular size, which could be due to differences in the degree and type of glycosylation. Glycosylation is known to vary with the tissue of protein expression (Rademacher et al., 1988). In the case of native BSSL, it has been even suggested (Hernell and Bläckberg, 1994) that the degree and type of glycosylation could vary among individuals and a month of lactation. Moreover, a difference in glycosylation between a native and recombinant BSSL was recently reported (Strömqvist et al., 1995).

We describe here the structural determination of the N-glycans isolated from 'averaged" BSSL (Bläckberg, et al., 1995). This structural characterization has been enabled through the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in conjunction with the action of specific exoglycosidases. The unique capabilities of MALDI mass spectrometry (MS) in carbohydrate analysis have gradually become evident during the recent years (Mock et al., 1991; Stahl et al., 1991; Harvey et al., 1994). The use of tandem mass spectrometry (MS/MS) has further made it feasible to analyze mixtures of neutral and sialylated oligosaccharides in one spectrum (Powell and Harvey, 1996).

In the work described here, the BSSL sample was first treated enzymatically to release N-linked oligosaccharides. These were further profiled for their approximate number and type by the established technique of high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and a newer technique of capillary electrophoresis (CE) using laser-induced fluorescence. Methylation of the sialic acid groups in the mixture of enzymatically released glycans, followed by MALDI-MS, revealed the existence of four main oligosaccharide components. The oligosaccharides were further sequenced using tandem MS (MS/MS) (Mechref et al., in press) with collision-induced dissociation (CID). Finally, five specific exoglycosidases were employed to confirm the sequences and yield the linkage forms of component monosaccharides.

Results and discussion

HPAEC-PAD and CE separations: orientation experiments

In order to assess the glycoprotein's complexity and the extent of sialylation, we first used HPAEC-PAD (Figure 1) under the chromatographic conditions conducive to the separation of multiply sialylated N-glycans of bovine fetuin, a 'standard glycoprotein" that has been investigated extensively (Green et al., 1988; Cumming et al., 1989). The retention times of the differently sialylated N-glycans of bovine fetuin are indicated in Figure 1a by horizontal brackets. A comparison of the retention times of the N-glycans of bovine fetuin and those of BSSL indicates that the N-glycans derived from BSSL are either neutral or monosialylated oligosaccharides. Moreover, one of the peaks appearing due to BSSL seemed to have the same retention time as one of the peaks due to fetuin. The extent of BSSL glycan complexity was further verified by capillary electrophoresis and MALDI mass spectrometry of the oligosaccharide mixture (see below).


Figure 1. (a) HPAEC-PAD map of the N-glycans derived from BSSL(the utilized elution program is summarized in Table II), and(b) electropherogram of the ANTS-labeled BSSL N-glycans. Conditions: background electrolyte, 25 mM sodium phosphate, pH 2.5; capillary, 50 cm/80 cm × 50 µm i.d. fused-silica capillary; voltage, 20 kV. Peaks marked with arrows are due to the reagent blank while those marked with asterisks correspond to the labeled N-glycans.

The electrophoretic map of the fluorescently labeled BSSL oligosaccharides is shown in Figure 1b. The electropherogram reveals the presence of several oligosaccharides: one major and two minor structures (labeled with asterisks). Although the separation and detection principles involved in Figure 1a vs. Figure 1b are different, there is a general agreement on the mixture complexity. The separation depicted in Figure 1b was performed in an acidic (pH 2.5) background electrolyte where any charges on the sialic acid groups were suppressed. Here, the separation is primarily based on the differences in structure rather than charge.

MALDI/MS of esterified and desialylated BSSL and fetuin N-glycans

A major problem encountered in the MALDI-MS of acidic oligosaccharides in the positive-ion mode is the loss of sialic acids and detection of multiple peaks resulting from the ion formation between the free acid and the salts. This fragmentation is known to decrease sensitivity of the MALDI/MS analysis for acidic oligosaccharides to the extent that, in some cases, the signal is totally lost. This problem was recently overcome by Powell and Harvey (Powell and Harvey, 1996) who acquired MALDI spectra of acidic oligosaccharides and gangliosides after methylation through the procedure of Norgard-Sumnicht et al. (Norgard-Sumnicht et al., 1995). This esterification step permits analysis of mixtures of neutral and acidic oligosaccharides in a single spectrum.

The MALDI mass spectrum of esterified N-linked oligosaccharides from BSSL is illustrated in Figure 2a. Four m/z signals were observed and tentative structures have been assigned, as based on these m/z values and the known structures of N-linked oligosaccharides of other glycoproteins (Green et al., 1988) that may have the same m/z values. The major peak at m/z 1969.5 was also observed in the MALDI spectrum of fetuin N-glycans (data not shown) where it corresponded to a known monosialylated biantennary oligosaccharide. This is also in agreement with the fact that one of the oligosaccharides derived from both BSSL and fetuin had the same retention time in HPAEC (Figure 1a), suggesting that both oligosaccharides have the same structure. Therefore, the signal observed at m/z 1663.5, differing from the signal observed at m/z 1969.5 by 306 (methylated sialic acid moiety) should belong to a desialylated biantennary structure. Desialylation of the BSSL oligosaccharides yielded a MALDI spectrum with m/z 1664.6 and 1810.6 (Figure 2b). On the other hand, the signal observed at m/z 2116.6 was believed to correspond to a fucosylated monosialylated biantennary structure while that at m/z 1809.2, which differed by 307 m/z units from the 2116.6 signal, was believed to correspond to a fucosylated desialylated biantennary structure. This information was deduced from the difference between these signals and those of the monosialylated dibranched structures, which was ~146, corresponding to a deoxyhexose residue (fucose). The MALDI mass spectra of the desialylated BSSL and fetuin oligosaccharides were acquired without the need to desalt the digestion mixture. This was made possible through the use of a new matrix (Chen et al., 1997) which is partially water-soluble, permitting to wash a spot on the MALDI plate with few drops of ice-cold water prior to the analysis.


Figure 2. MALDI mass spectra of esterified (a) and desialylated (b) N-glycans derived from BSSL. The m/z values correspond to monoisotopic masses of the sodiated ions. The brackets around the structures symbolize their tentative nature. Symbols: solid circles, mannose; open squares, N-acetylglucosamine; open squares, galactose; open triangles, methylated sialic acid; solid circles, fucose.

CID-MALDI/MS of desialylated glycans

An attractive feature of a MALDI time-of-flight instrument with the reflectron geometry is its ability to provide the means of fragmentation for the analyzed oligosaccharides. In principle, this leads to the determination of sequence, branching and linkage forms (Spengler et al., 1994; Harvey et al., 1995; Lemoine et al., 1996; Penn et al., 1996; Mechref et al., in press). We have utilized this capability in providing the structural information regarding the sequence and branching in the two desialylated structures observed in Figure 2b.

The mass spectrum obtained through the collision-induced dissociation (due to the parent m/z 1663) is illustrated in Figure 3. There are three sets of fragments observed in this spectrum, corresponding to the B, Y, and combined B and Y fragments according to the nomenclature of Domon and Costello (Domon and Costello, 1988). From the fragments observed in this spectrum, it can be determined that this structure corresponds to a biantennary complex oligosaccharide. Moreover, the CID spectrum shown in Figure 3 resembled that of the biantennary structure from bovine fetuin (data not shown). While the fragmentation pattern was helpful in assigning the structure, the absence of any cross-ring fragmentation limited the determination of linkage forms.


Figure 3. CID-MALDI mass spectrum of m/z 1663 as the parent ion. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated fragments.

The information deduced from the CID spectrum acquired for the m/z 1809 was even more revealing (Figure 4). Again, three sets of fragments were observed, corresponding to B, Y, and combined B and Y fragments. The observed fragments provided information regarding the monosaccharide residue sequence and branching, but more importantly, limited the linkage of the fucose residue to the N-acetylglucosamine residue at the reducing-end. This was determined by the presence of Y4x/Y4x signal (m/z 1076.87), which is a fragment lacking the N-acetylglucosamine residues on the nonreducing end, and by the presence of B5 signal (m/z 1443) corresponding to a fragment resulting from a loss of the reducing-end N-acetylglucosamine residue and a fucose residue (Figure 4). This finding will further be supported by the work with enzymatic cleavages (see below).


Figure 4. CID-MALDI mass spectra of m/z 1809. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated fragments.

To this point, we have determined the sequence and branching of the N-glycans derived from BSSL, but the absence of any cross-ring fragmentation in the CID spectra limited the determination of linkages. Fortunately, this information could be obtained through sequential enzymatic cleavages of the N-glycans using specific exoglycosidases.

Enzymatic determination of sequence and linkages

Table I lists the enzymes that have been employed in verification of the monosaccharide sequences and determination of the linkages in BSSL oligosaccharides. A negative sign in this table indicates that no change in a MALDI spectrum was observed after the enzymatic digestion. The sialic acid linkage in the oligosaccharide structures was determined to be an [alpha]-2,6-type, as concluded from a negative action of the neuraminidase that is specific for cleavage at [alpha]-2,3-position, and a positive action of the neuraminidase cleaving both [alpha]-2,3- and [alpha]-2,6-linkages (Figure 2b).

Table I. Specificity of selected exoglycosidases and their action on BSSL N-glycans
Exoglycosidase Specificity Digestion of BSSL oligosaccharides
[beta]-d-galactosidase from Diplococcus pneumoniae Hydrolyzes terminal galactose residues which are [beta]-1,4-linked to GlcNAc. +
N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae Cleaves terminal N-acetylglucosamine residues that are [beta]-linked to oligosaccharides. +
[alpha]-1,3/4-fucosidase from Streptomyces species Cleaves [alpha]-1,3- and [alpha]-1,4-linked terminal fucose on N-linked oligosaccharides. -
neuraminidase from Arthrobacter ureafaciens Hydrolyzes terminal N- or O-acylneuraminic acids which are [alpha]-2,3- and [alpha]-2,6-linked to galactose, Hex, GlcNAc or N- or O-acetyl neuraminyl residues in oligosaccharides or colominic acid. +
neuraminidase from Newcastle disease virus Hitchner B1 strain Hydrolyzes very specifically terminal [alpha]-2,3-bound neuraminic acids of N- or O-glycosidically linked oligosaccharide chains in glycoproteins and glycolipids. -
+, Symbolizes occurrence of a cleavage.
-, Symbolizes absence of a cleavage.

Incubation of the BSSL N-glycans with neuraminidase and [beta]-galactosidase caused a shift of ~324 m/z for the two signals observed upon a treatment of the N-glycans with neuraminidase alone (Figures 2b and 5b). This shift can only be due to the loss of two terminal galactose residues. Therefore, the terminal hexose present in the structures was judged to be that of a galactose linked to the adjacent monosaccharide through a [beta]-1,4 linkage.

Treatment of the BSSL oligosaccharides with N-acetylglucosaminidase after desialylation and degalactosylation resulted in a shift of ~404 m/z , in addition to the 324 m/z shift observed from degalactosylation. This additional shift is due to the loss of two N-acetylglucosamine residues which are [beta]-1,2-linked (Figures 2b and 5c). For the signal at m/z 1809, the shift of 404 m/z corresponding to a loss of just two N-acetylglucosamines (due to a treatment with N-acetylglucosaminidase) suggested the site of fucosylation to be on either of the two reducing-end GlcNAc residues. This supports the CID fragmentation pattern described above.

Moreover, the site of fucosylation was judged to be on the reducing-end GlcNAc, as based on the action of endoglycosidase F which cleaves specifically the N-linked oligosaccharides between the two reducing-end GlcNAc residues. The MALDI spectrum of desialylated BSSL oligosaccharides cleaved by endoglycosidase F showed a signal at m/z 1460.1, corresponding to a dibranched structure with one GlcNAc at the reducing end (Figure 5d). Linkage of the fucose residue to the reducing-end GlcNAc was found to be of an [alpha]-1,6-type, as deduced from the negative results observed with [alpha]-1,3/4-fucosidase (see Table I and Figure 5a).

Scheme 1 summarizes the structures of BSSL N-glycans as based on our determinations. The linkage and sequence of the first five monosaccharide residues were not determined enzymatically, since it is widely known that N-linked oligosaccharides generally contain a common pentasaccharide core consisting of three mannoses and two GlcNAc residues (the so-called 'mannose core").


Scheme 1. Structures of BSSL N-glycans.


Figure 5. MALDI mass spectra of the exoglycosidase digests. (a) Treatment with neuraminidase and [alpha]-1,3/4-fucosidase; (b) treatment with [beta]-galactosidase; (c) treatment with N-acetylglucosaminidase; (d) desialylated BSSL oligosaccharides cleaved by Endoglycosidase F. Symbols as in Figure 2. The m/z values correspond to monoisotopic masses of the sodiated ions.

The structures elucidated here are similar to those reported for another human bile-salt stimulated lipase that was isolated from pancreatic juice (Mas et al., 1993; Sugo et al., 1993). However, the N-glycans from the pancreatic lipase were more heterogeneous and their fucosylation was determined to be [alpha](1-6)- and [alpha](1-2)-types, linked to the innermost N-acetylglucosamine residue and a terminal galactose residue, respectively. Additionally, the structural determinations reported here are in general agreement with the very recent general studies (Landberg et al., 1997) aiming at comparison of the native and recombinant forms of the human milk BSSL.

Materials and methods

Fetuin from the fetal calf serum and neuraminidase (EC 3.2.1.18) from Arthrobacter ureafaciens were purchased from Sigma Chemical Co. (St. Louis, MO). The BSSL sample (from a pool of breast milk of over 100 individuals) was received from Astra Hässle AB (Umeå, Sweden), after being prepared according to the procedure reported by Bläckberg et al. (Bläckberg et al., 1995). N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum, a recombinant product from E.coli (EC 3.2.2.18), endoglycosidase F from Flavobacterium meningosepticum (EC 3.2.196), N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae (EC 3.2.1.30), [beta]-galactosidase from Diplococcus pneumoniae (EC 3.2.1.23), neuraminidase (Sialidase) from Newcastle disease virus (EC 3.2.118) and [alpha]-1,3/4-fucosidase from Streptomyces species (EC 3.2.1.51) were all from Boehringer Mannheim (Indianapolis, IN). All common chemicals were received from Aldrich (Milwaukee, WI). The fluorescence labeling reagent, 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS), was purchased from Molecular Probes, Inc. (Eugene, OR).

Enzymatic cleavages

The N-linked oligosaccharides from bovine fetuin and BSSL were enzymatically released by PNGase F. The glycoproteins to be digested were reconstituted in 0.05 M sodium phosphate buffer (pH 7.5), followed by addition of PNGase F (2.0 U/1.0 mg glycoprotein). The samples were subsequently incubated for 24 h at 37°C.

The enzymatically released oligosaccharides were recovered by applying the digestion mixtures to C18 Sep-Pak cartridges (Waters Corp., Milford, MA) that were preconditioned with methanol, acetonitrile, and aqueous methanol (1:4 v/v methanol/water). The oligosaccharides were eluted with 3 ml of the aqueous methanol solution and then dried under a stream of nitrogen. In the case of endoglycosidase F, the BSSL sample (100 µg) was dissolved in 20 µl of 50 mM sodium acetate buffer (pH 6.0) and incubated with 100 mU of the enzyme at 37°C for 18 h. The oligosaccharides cleaved were eventually pooled as described above. Specificities of the exoglycosidases used in this study are summarized in Table I. All digestions were performed on the oligosaccharides released from 100 µg of BSSL. Monosaccharides were removed sequentially from the nonreducing end of BSSL oligosaccharides by incubation with specific exoglycosidases at different times.

Desialylation. The sialic acid residues of the N-glycans derived from fetuin and BSSL were liberated by reconstitution of the oligosaccharides released from 100 µg of a glycoprotein in 50 mM sodium phosphate buffer (pH 6.0), followed by the addition of neuraminidase from Arthrobacter ureafaciens, or Newcastle disease virus (~20 mU/ 1.0 nmol of substrate) and incubation for 12 h at 37°C.

Degalactosylation. The samples desialylated, as described above, were treated with 20 mU [beta]-galactosidase from Diplococcus pneumoniae (in the same buffer) for 18 h at 37°C.

Removal of N-acetylglucosamine. The desialylated and degalactosylated sample was desalted by passing through a fritted 1 ml syringe containing mixed DOWEX-50W (H+) and DOWEX 1 (OH-) resins. The eluent was lyophilized before reconstitution in 50 mM sodium citrate/phosphate buffer (pH 5.0) and incubation with 20 mU of N-acetyl-[beta]-d-glucosaminidase from Diplococcus pneumoniae for 18 h at 37°C.

Treatment with [alpha]-1,3/4-fucosidase from Streptomyces species. BSSL oligosaccharides were treated with 20 mU neuraminidase and 20 mU [alpha]-1,3/4-fucosidase in 50 mM sodium phosphate buffer (pH 6.0), at 37°C for 18 h. In all cases, the enzymatic activity was stopped by heating the reaction microtubes for 5 min at 100°C prior to a mass-spectrometric analysis.

Esterification of acidic oligosaccharides

The sialic acid residues of acidic oligosaccharides were esterified according to the procedure described by Powell and Harvey (Powell and Harvey, 1996). Briefly, the acidic oligosaccharides dissolved in water were applied to a short column (made from a fritted 10 ml plastic syringe) containing Dowex-50W resin (Sigma Chemical Co.) that had been preconditioned with 1.0 M sodium hydroxide and water. The eluent was then lyophilized and dissolved in dry dimethylsulfoxide (DMSO). Methyl iodide was added, and the mixture was thoroughly mixed and allowed to react at room temperature for 2 h. Finally, the reaction mixture was dried under a stream of nitrogen and desalted by passing through a short desalting column (made from a fritted 10 ml plastic syringe) containing a mixture of cation- and anion-exchange resins. The eluent was lyophilized and redissolved in deionized water.

Labeling of BSSL N-glycans

BSSL oligosaccharides were labeled with ANTS through reductive amination. The oligosaccharides, cleaved from 75 µg of BSSL, were dissolved in 2 µl 0.15 M ANTS in 3:17 propionic acid:DMSO and 2 µl 1.0 M sodium cyanoborohydride in DMSO. The reaction mixture was vortexed prior to incubation in a water bath at 40°C for 15 h. Propionic acid was employed to eliminate or minimize hydrolysis of the sialic acid groups. Prior to a CE analysis, the sample was diluted 100-fold with deionized water.

Sample and matrix preparation for MALDI/MS studies

Arabinosazone, a new matrix reported recently by our laboratory (Chen et al., 1997) was used exclusively for all MALDI/MS measurements. Esterified samples as well as the exoglycosidase digests were prepared for the MALDI analysis by spotting 1.0 µl of the test solution on a polished stainless-steel sample plate, followed by addition of 1.0 µl arabinosazone dissolved in ethanol/water (3:1 v/v) at a final concentration of 10 mg/ml and allowing the spot to dry at room temperature.

Instrumentation

MALDI-Time-of-Flight Mass Spectrometry. Mass spectra were acquired on a Voyager-DE RP Biospectrometry Workstation instrument (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). The instrument operates both in the reflector and linear modes of detection and has the delay extraction capability. MALDI spectra were acquired at 25 kV accelerating voltage, while the low-mass gate was used to discard the ions with m/z values of less than 500. All acquired spectra were smoothed by applying a 19-point Savitzky-Golay smoothing routine (Savitzky and Golay, 1964). The instrument is equipped with a collision cell, with argon being used as the collision gas. For collision-induced dissociation (CID) spectra, the precursor ion was selected by the time ion selector, with a mass window of approximately 45 m/z. The CID spectra were acquired at 25 kV accelerating voltage, while the reflectron voltage was successively decreased by 10%, allowing the detection of certain fragments at a time. Generally, 10-12 segments were acquired at 100 scans for each segment, and the composite CID spectrum was a combination of all acquired segments, as generated by the instrument software.

HPAEC-PAD instrument and separation conditions. HPAEC-PAD mapping was performed using the Dionex Bio-LC System (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-100 column (4.6 × 250 mm) working at a flow rate of 1 ml/min at ambient temperature. The pulse potentials and durations were 0.05 V/300 ms, 0.75 V/120 ms, and -0.70 V/60 ms, working at 300 nA full scale. The gradient utilized for acquiring the map is summarized in Table II.

Capillary electrophoresis. The instrument for capillary electrophoresis was assembled in-house (Sudor and Novotny, 1995) from the commercially available components. It utilized a high-voltage power supply (0-40 kV) from Spellman High Voltage Electronics (Plainview, NJ). A 325 nm helium-cadmium laser, model 56X from Omnichrome (Chino, CA) was used as a light source. A 600 µm fiber-optic placed at right angle to the incident laser beam was employed to collect fluorescence emission. A long-pass, low fluorescence emission filter (cutoff > 360 nm) isolated the signal which was monitored using R928 photomultiplier tube (Hamamatsu Photonics K.K., Shizuoka Prefecture, Japan) and amplified with model 128A lock-in amplifier (EG&G Princeton Applied Research, Princeton, NJ). A 50 cm (effective length)/80 cm (total length) fused silica capillary with 50 µm I.D. (Polymicro Technologies, Phoenix, AZ) was used as the separation column. 20 kV applied voltage was used to separate the ANTS-labeled BSSL N-glycans.

Table II. Gradient utilized for obtaining HPAEC-PAD maps
Min Eluent 1 (250 mM sodium hydroxide) Eluent 2 (500 mM sodium acetate) Eluent 3 (Deionized water)
0 40% 0% 60%
10 40% 0% 60%
60 40% 50% 10%
70 40% 50% 10%

Acknowledgments

We thank Andrew Baker for his assistance with mass-spectrometric instrumentation, and Drs. Jörgen Vessman and Lennart Lundberg for their interest in this study. This study was supported by Grant GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services, and a grant-in-aid from Astra/Hässle, Mölndal, Sweden.

Abbreviations

ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; BSSL, bile salt-stimulated lipase; CE, capillary electrophoresis; CID, collision-induced dissociation; DMSO, dimethylsulfoxide; GlcNAc, N-acetylglucosamine; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PNGaseF, N-glycosidase F.

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1To whom correspondence should be addressed.
2Present address: Department of Chemistry, University of the United Arab Emirates,Al-Ain, United Arab Emirates


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