Institute of Biological Chemistry, Academia Sinica, Institute of Biochemical Sciences, National Taiwan University, and the 2Department and Institute of Parasitology, National Yang-Ming University, Taipei, Taiwan R.O.C.
Received on July 18, 2000; revised on September 18, 2000; accepted on September 21, 2000.
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Abstract |
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Key words: Schistosoma mansoni/Schistosoma japonicum/ N-glycosylation/mass spectrometry/structural analysis
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Introduction |
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Previous work based primarily on S. mansoni (reviewed in Cummings and Nyame, 1996; 1999), have demonstrated that a highly immunogenic, uniquely multifucosylated terminal sequence carried on complex O-glycans specifically characterizes the glycocalyx of the invading cercariae (Khoo et al., 1995
) which is rapidly shed on successful penetration of host skins. The ensuing schistosomula and adult worm stages apparently adopt a glycosylation profile rather similar to that of the mammalian host in which they reside. The major N-glycans were shown to consist of both high mannose and complex types of biantennary structures terminating with the LacdiNAc (GalNAcß1
4GlcNAcß1
) sequence (Srivatsan et al., 1992b
) or tri- and tetraantennary structures with poly-N-acetyllactosamine sequence containing the Lewis X (Lex) antigen (Srivatsan et al., 1992a
), but no sialic acid. With the onset of egg laying, the immunogenic multifucosylated structures recur on the egg glycosphingolipids and total glycoprotein extracts, which are probably synthesized by the developing miracidium inside the egg. Comparative studies on the egg glycans from S. japonicum (Khoo et al., 1997b
), which infects a very different intermediate snail host, revealed a substantially distinct profile from that of S. mansoni. Notably, a portion of the N-glycans from S. mansoni eggs was shown to be based on a xylosylated,
6-fucosylated trimannosyl core, whereas a portion of those from S. japonicum contains a xylosylated
3-,
6-difucosylated core.
Core ß2-xylosylation and/or 3-fucosylation of N-glycans is largely confined to plants (Lerouge et al., 1998
; Wilson et al., 1998
) and rare examples in parasitic helminths (Haslam et al., 1996
; Khoo et al., 1997b
; van Die et al., 1999
), insects (Altmann et al., 1999
), and mollusc hemocyanins (Kamerling and Vliegenthart, 1997
) but not known to occur in mammals. Instead, these have been implicated as allergenic epitopes (Tretter et al., 1993
; Garcia-Casado et al., 1996
; van Ree et al., 2000
), probably contributing to host IgE response on parasitic helminth infection (van Die et al., 1999
). Our identification of such schistosomal species-specific core modification in their egg glycoprotein extracts prompted us to examine the N-glycosylation profiles of other developmental stages of both species, so as to fully understand their immunobiological significance in hostparasite interactions. We report here full structural characterization of core xylosylated N-glycans from S. mansoni cercariae that carry terminal Lex as the predominant terminal epitopes. In comparison, the corresponding Lex-carrying N-glycans from S. japonicum cercariae appear to lack xylosylation or core
3-fucosylation. The unexpected high abundance of Lex expression in the cercariae relative to adult worms provides a new insight into its possible functional roles not previously appreciated.
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Results |
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The Lex structure could also be indirectly conjugated to the core via another GlcNAc residue, which could itself be fucosylated as found in N11.7 and N12.8. The strong fragment ion signals at m/z 1057 and 638 but not 434, as afforded by CID-MS/MS analysis of N12.8 (Figure 3D) indicated that one antennae was Hex-(Fuc)HexNAc-(Fuc)HexNAc, whereas the other was Hex-(Fuc)HexNAc. Any one of the three fucoses could be missing in N11.7, giving rise to all possible combination of fragment ion signals observed (Figure 3C). Only 3,4-linked GlcNAc and 4-linked GlcNAc and no GalNAc were detected by linkage analysis of both components (Table I). On 3,4-fucosidase digestion, two Fuc residues were removed from N12.8, which rendered the product susceptible to further ß4-specific galactosidase digestion. The final product obtained was shown by MS analysis to correspond to Fuc2Xyl1HexNAc5Hex3. In comparison with the linkage analysis data prior to glycosidase digestions, terminal GlcNAc was detected concomitant with a reduction in intensity of the 3,4-linked GlcNAc peak. It was thus concluded that the initial
3,4-fucosidase digestion resulted only in the removal of the Fuc on the Lex structure, whereas the inner Fuc was not affected. Two very minor components eluting later than 13 Glc units on normal phase HPLC were detected to have additional HexNAc and Fuc1HexNAc1, respectively, as compared to N12.8, which suggested that the biantennary core structure could have both antenna extended by this unusual Gal-(Fuc)GlcNAc-(Fuc)GlcNAc sequence although further confirmatory analysis was prevented by the sample amount available.
Other minor components in S. mansoni cercarial N-glycans
Other minor structures were evidently present (Figure 1), some made more apparent after enzymatic treatments (Figure 2) or HPLC fractionation (Figure 5). Notably, the presence of LacdiNAc and fucosylated LacdiNAc type of structures were implicated by the fragment ions at m/z 260 (HexNAc+), 505 (HexNAc2+), and 679 (Fuc1HexNAc2+). The presence of m/z 434 (Fuc1HexNAc+) and 679 but not 853 (Fuc2HexNAc2+) indicated that the HexNAc-HexNAc- terminus can be mono-fucosylated at either HexNAc residue but not both. Alternatively, an incompletely extended terminal HexNAc residue may be directly fucosylated to yield the observed Fuc1HexNAc+ fragment ion. In support of these other minor forms is the presence of terminal GlcNAc, terminal GalNAc, and 3-linked GalNAc in the linkage analysis (Figure 4, inset) along with the three most abundant HexNAc residues, namely, 4-linked GlcNAc, 3,4-linked GlcNAc, and 4,6-linked GlcNAc. After sequential enzyme treatment of the total N-glycans with a combination of -fucosidases and ß-galactosidase, the major resistant core structures could be assigned as ±Xyl (Man3GlcNAc2) with one, two, three, four, or no additional HexNAcs attached (Figure 2C). CID-MS/MS analysis on [M+H]+ of HexNAc2(Xyl1Man3GlcNAc2) at m/z 1799 and HexNAc3(Xyl1Man3GlcNAc2) at m/z 2044 showed the presence of HexNAc-HexNAc+ sequence (m/z 505) in both. The absence of a HexNAc3+ ion in the latter indicated that one single HexNAc and one HexNAc2 element were extending from the two antenna of a biantennary structure. It remains possible that additional triantennary structure was present but not a monoantennary one with a stretch of three HexNAcs.
The major N-glycan profile of S. japonicum cercariae
The lack of sample material prevented a full characterization of the S. japonicum cercarial N-glycans. However, FAB-MS analysis of the permethyl derivatives revealed some interesting features in comparison with those of S. mansoni. As shown in Figure 6, none of the major components detected appeared to carry xylosylation. Apart from high mannose-type structures, the other major [M+Na]+ molecular ions afforded could be assigned as (i) core fucosylated Hex2-5HexNAc2 (m/z 1141, 1345, 1549, and 1753), likely to be truncated pauci mannose-type; and (ii) fucosylated trimannosyl core extended with a single HexNAc (m/z 1590), a single Hex-HexNAc (m/z 1794), a single Fuc1Hex1HexNAc (m/z 1968), two Hex1HexNAc1 units (m/z 2244), and two Fuc1Hex1HexNAc1 units (m/z 2592). Assignment of core 6-fucosylation was supported by susceptibility to N-glycosidase F and bovine kidney
-fucosidase. Digestion with the latter enzyme resulted in a shift of the molecular ion signals to m/z values corresponding to loss of a Fuc residue (data not shown). A-type oxonium fragment ions at m/z 1495 and 2118, which were derived from cleavage at the chitobiose core of [Fuc1Hex1HexNAc1]1[Hex3HexNAc2Fuc] ([M+Na]+ at m/z 1968) and [Fuc1Hex1HexNAc1]2[Hex3Hex-NAc2Fuc] ([M+Na]+ at m/z 2592), further indicated that one Fuc was attached to the reducing end HexNAc of these two species. CID-MS/MS analysis on the [M+H]+ parent ion of the former yielded major daughter ions at m/z 196, 432, 450, 638, 810, and 1495, which, except for the last ion, were identical to those afforded by the Lex containing structures from S. mansoni described above (Figure 3AB). On treatment with
3/4-specific fucosidase, one Fuc was removed from this species. The CID-MS/MS analysis of the digestion product unambiguously showed that daughter ions at m/z 638, 810, and 1495 were replaced by new signals at m/z 464, 636, and 1321, as would be expected from removal of the Fuc 3-linked to GlcNAc in Lex. Our data therefore strongly suggested that the major N-glycans of S. japonicum cercariae resembled those of S. mansoni by carrying core
6-fucosylation and terminal Lex epitopes but were not core xylosylated.
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To further ascertain if Lex could be found among the complex- and hybrid-type structures, the N-glycans from S. mansoni adults were pyridylaminated and fractionated on normal phase HPLC (Figure 8). The fluorescence-detected peaks were collected, screened by MS, and, where sample materials were sufficient, sequentially digested with exo-glycosidase and/or subjected to CID-MS/MS to confirm their terminal structures, as summarized in Table IV. The assignment of high mannose structures in N6.7, N7.7, N8.7, N9.6, and N10.4, pauci mannose and other truncated cores, and monoantennary structures in N6.9 and N7.7 were supported by their respective susceptibility to -mannosidase trimming. The presence of additional Fuc, which may constitute Lex and fucosylated LacdiNAc in the monoantennary and complex-type structures, were indicated by their molecular composition. Treatment with bovine kidney
-fucosidase in general only removed one Fuc corresponding to the core
6-Fuc, whereas additional Fuc on the antenna were only susceptible to
3,4-fucosidase. CID-MS/MS on the parent ions at m/z 2038 and 2079 in N7.7 yielded the characteristic sets of daughter ions attributable to terminal Hex-4(Fuc-3)HexNAc (m/z 432, 638) and terminal HexNAc-4(Fuc-3)HexNAc (m/z 260, 473, 679) structures, respectively. For N6.9, which was of higher abundance, further reverse phase HPLC on the
-fucosidase treated sample yielded four major peaks (data not shown). An
-mannosidase resistant peak was digested by ß-GlcNAcase to give the trimannosyl core, indicating a GlcNAcß1
6(GlcNAc-ß1
3)Man3GlcNAc2 structure consistent with its defined composition. The remaining three peaks were shown to be monoantennary by their susceptibility to
-mannosidase, which removed one Man residue from each. Based on their molecular composition, reverse phase elution order and Glc unit difference (Tomiya and Takahashi, 1998
), the earliest eluting peak was deduced to be a mixture carrying either a LacNAc or LacdiNAc on the 3-arm, whereas the other two later eluting peaks carried a LacNAc and a LacdiNAc on the 6-arm, respectively. The LacNAc containing components were converted to the expected core by sequential treatment with ß-galactosidase and then ß-GlcNAcase, whereas the LacdiNAc components were only susceptible to the nonspecific ß-HexNAcase and not the specific ß-GlcNAcase. Based on these analyses, other larger N-glycan components from the adult worms were likewise implicated as complex types extended with LacNAc and/or LacdiNAc units, some of which were fucosylated. The smallest N-glycans with a Lex determinant as shown here were the monoantennary type with a single Lex unit on an
6-fucosylated but not xylosylated trimannosyl N,N'-diacetylchitobiose core.
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Discussion |
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The schistosomal egg glycans represent the first and only example in animal glycoproteins where both core 3-fucosylation and ß2-xylosylation were chemically shown to occur on the N-glycans (Khoo et al., 1997b
). Unlike plant glycoproteins, these modifications almost invariably occur on an
6-fucosylated core. As characterized previously (Khoo et al., 1997b
), either core
3-fucosylation or xylosylation can occur on the egg N-glycans of both schistosome species, but coexistence of both core modifications appeared to be restricted to S. japonicum. The detection of core difucosylated and xylosylated N-glycans is therefore diagnostic of S. japonicum and possibly is also developmental stagespecific, restricted to the eggs and miracidia. We have not been able to detect such core modification on the N-glycans from any other S. japonicum developmental stages examined, including the cercariae, schistosomula, and adults. In contrast, ß2-xylosylation is a prominent structural feature of S. mansoni cercarial N-glycans but down-regulated as the parasite develops into adult. A recent study based on Western blot analysis with core
3Fuc- and ß2Xyl-specific antibodies (van Die et al., 1999
) has indicated that ß2-core xylosylation can be found on the adult extracts of S. mansoni but not S. japonicum. Although we failed to detect core xylosylation in the adults of either species, we did provide chemical evidence that core xylosylation is characteristic of S. mansoni cercariae. It is possible that residual level of expression on the adults may still be detectable by antibodies. On the other hand, the implicated presence of
3-core fucosylation on the adults (van Die et al., 1999
) could not be substantiated by our mass spectrometry analysis of N-glycosidase Areleased N-glycan samples.
Apart from N-glycan core fucosylation, at least two other antigenic fucosylated epitopes feature prominently on schistosomal glycans. The first is the unique multifucosylated HexNAc termini with internal Fuc residue, ±Fuc2Fuc
3GalNAcß(±Fuc
2Fuc
2Fuc
3)4GlcNAc-, as found on the complex O-glycans of S. mansoni cercarial glycocalyx (Khoo et al., 1995
), which probably constitute the epitope recognized by a mAb raised against cercarial glycoproteins (Dalton et al., 1987
). Immunolocalization with this mAb showed that this fucosylated epitope was expressed on the surface of cercariae and newly transformed schistosomula but not detectable on the adult worms (Köster and Strand, 1994
). Its recurrence on the egg antigens has been indicated by immuno-cross-reactivities (Weiss and Strand, 1985
; Weiss et al., 1986
) and further corroborated by structural studies that identified similar nonreducing terminal epitopes on the egg glycoproteins and glycolipids (Levery et al., 1992
; Khoo et al., 1997a
,b). There are potentially many structural variations to this epitope. In its simplest form without any of the Fuc, this terminal disaccharide corresponds to the so-called LacdiNAc sequence (GalNAcß4GlcNAcß-). Both LacdiNAc and its fucosylated version, GalNAcß(Fuc
3)4GlcNAcß-, were readily detectable on the glycans from each of the developmental stages examined to date (Cummings and Nyame, 1999
), as well as those characterized here.
Another fucosylated epitope as defined by mAb raised against S. mansoni egg glycoproteins has been characterized to recognize Lex structure (Ko et al., 1990; Köster and Strand, 1994
). This mAb or mAb against Lex were shown to bind to the surface of schistosomula and adults and the extracts from adults of both S. mansoni and S. japonicum (Nyame et al., 1998
). Structurally, Lex has been defined to be present on the S. mansoni adults (Srivatsan et al., 1992a
), the egg glycoproteins (Khoo et al., 1997b
), as well as the O-glycans from adult gut-associated excretory circulating cathodic antigen (CCA) (van Dam et al., 1994
). Because the Lex-recognizing mAb failed to bind to the surface of S. mansoni cercariae (Köster and Strand, 1994
), it was thought that the expression of Lex is developmentally regulated and only initiated after transformation into schistosomula, just as the multifucosylated glycocalyx is shed. However, data (including this study) have indicated otherwise.
First, the same mAb that failed to bind to the surface of the cercariae has actually been shown to bind to the cercarial and egg extracts more readily than to the adult extracts (Weiss and Strand, 1985). Second, antibody that recognized Lex was among the humoral response elicited in mice vaccinated with irradiated cercariae of S. mansoni (Richter et al., 1996
). Both studies argued that Lex epitope was presented by S. mansoni cercariae. More recently, the glycosphingolipids from S. mansoni cercariae were characterized to be dominated by Lex and pseudo Ley structures (Wuhrer et al., 2000
). Our own research as reported here indicates that high level of Lex was indeed present on the cercarial N-glycans as well as O-glycans (unpublished data), even more so than on adult glycans on a comparative basis. Thus, the cercariae stage does express an abundance of Lex on a variety of glycoconjugates. However, it is possible that surface exposure of Lex may be masked by the more immunodominant glycocalyx multifucosylated glycans. Instead, the anti-Lex mAb was shown to recognize secreted material near the acetabular gland openings and in the ventral sucker of S. mansoni cercariae (Köster and Strand, 1994
; van Remoortere et al., 2000
). We have shown that if the cercariae glycoprotein extracts were fractionated by Anguilla anguilla lectin affinity column, most of the Lex-containing glycoproteins characterized in this study were found in the flow-through fractions, distinct from the population that bound to the column that contained the previously characterized glycocalyx multifucosylated epitopes (Khoo et al., 1995
). Thus, the two different fucosylated epitopes may be carried on two different sources, and the Lex carrying glycoproteins may well be related to secreted materials, as they were very readily extracted. The high abundance of Lex epitopes found on cercariae would constitute an important first source of this immunogen following infection, well before secretion of CCA from the adults or the onset of egg laying.
Sera from humans and rodents infected with S. mansoni and S. japonicum have been shown to contain antibodies reactive with Lex (Nyame et al., 1996,1997,1998). Schistosomal infection is also known to induce a pronounced Th2-type response associated with significant IgE production. Because Lex has been shown to induce murine B-1 cells to secrete large amounts of IL-10 that down-regulate type 1 CD4+ T cells, it may function as an immune activator of Th2-associated responses (Velupillai and Harn, 1994
). Although most of the immunopathogenic activities of schistosomal glycans have been attributed to the egg antigens, it is of interest to note that the invading cercariae is itself an important source of Lex, in addition to presenting an immunopotent glycocalyx.
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Materials and methods |
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Extraction of glycoprotein and preparation of N-glycans
Collected schistosomal cercariae, schistosomula, and adult worm samples were homogenated by sonic treatment at 4°C, and lipids were removed by sequential extraction with 5:10:3 CHCl3:CH3OH:H2O and 30:60:8 CHCl3:CH3OH:0.4M sodium acetate (v/v/v). Delipidated homogenates were vortexed in 6 M guanidine hydrochloride, and the supernatants were dialyzed against running water at 4°C. Alternatively, cercarial glycoproteins were extracted by phenol/water partition method as described (Xu et al., 1994). Dialyzed and lyophilized glycoprotein extracts were then redissolved in 20 mM Tris buffer (pH 7.67.8, with 1 mM each of CaCl2, MgCl2, MnCl2, and 0.1 M NaCl) and loaded onto a lectin affinity column (Anguilla anguilla gel, EY laboratories) equilibrated in the same buffer. Nonbound glycoproteins were eluted with two column volumes of the same Tris buffer, and bound glycoproteins were subsequently eluted with 100 mM fucose in the same Tris buffer. Elution were monitored online by UV absorbance at 280 nm. Fractions collected were pooled accordingly, dialyzed against water, and then lyophilized. The majority of N-glycans was found on nonbound fractions, whereas the bound glycoprotein fractions contained mainly the multifucosylated O-glycans characterized previously (Khoo et al., 1995
), with a small amount of N-glycans similar in composition to those identified in the nonbound fraction.
The crude glycoprotein extracts obtained were first digested with nonTPCK-treated trypsin (Roche) (1:50 enzyme: protein ratio, w/w; 37°C for 4 h in 50 mM ammonium hydrogen carbonate buffer, pH 8.4) followed by nonTLCK-treated chymotrypsin in the same conditions. The digested glycopeptides were loaded onto C18 Sep-pak® cartridge (Waters). Glucan polymer and other hydrophilic contaminants were washed off with 5% aqueous acetic acid, and the bound peptides were eluted with a step gradient of 20, 40, and 60% 1-propanol in water. All the eluted fractions were pooled, dried down, and then incubated with N-glycosidase F (5 units, Roche) overnight at 37°C in 50 mM ammonium bicarbonate buffer, pH 8.4. Released N-glycans were separated from peptides/glycopeptides using the same C18 Sep-pak procedure. The pooled peptides/glycopeptides fractions were then digested with N-glycosidase A from almond (0.5 mU, Calbiochem) in 50 mM ammonium acetate buffer, pH 5, at 37°C overnight. N-glycans released by N-glycosidase A were likewise separated from the de-N-glycosylated peptides/glycopeptides by the same C18 Sep-pak procedures.
Fluorescent labeling and HPLC purification of N-linked glycans
The N-glycosidase Freleased N-glycans were reductively aminated with 2-aminopyridine at the reducing end exactly as described (Hase, 1994). Excess reagents were first removed by sequential co-evaporation with CH3OH:triethylamine (3:1, v/v), toluene, and toluene:methanol (2:1, v/v) at 50°C, followed by gel filtration chromatography on a Bio-Gel P-2 column (50 cm x 1.5 cm, equilibrated and eluted with 10 mM ammonium bicarbonate). Elution of PA-tagged glycans were monitored online by UV absorbance at 280 nm wavelength.
HPLC analysis was performed on a Hewlett Packard 1100 series LC equipped with a thermostatted column compartment and fluorescence detector. The PA-labeled glycans were size fractionated on a PalPak type N column (Takara, 4.6 x 250 mm) at a flow rate of 1 ml/min, 30°C, and detected by fluorescence with excitation and emission wavelengths set at 310 and 380 nm, respectively. Buffers A and B contain 25:75 and 50:50 stock buffer: acetonitrile (v/v), respectively, where stock buffer was aqueous solution containing 10% acetonitrile and 3% acetic acid, titrated to pH 7.3 with triethylamine. The column was equilibrated and maintained at 10% B for 5 min, then linearly increased to 100% B in 40 min and kept at 100% B for another 15 min. For second dimension separation or desalting after enzyme digestion, reverse phase HPLC was carried out on a Hypersil ODS column (5 µ, 4.0 x 125 mm, Hypersil), at a flow rate of 0.5 ml/min, 40°C, and detected by fluorescence at 320/400 nm excitation/emission wavelengths. Buffer A was 0.1 M ammonium acetate, pH 4.0; buffer B was 15% methanol in buffer A. Gradient used: 5% to 20% B at 15 min, to 35% B at 50 min, to 80% B at 60 min. In both modes, PA-labeled isomalto-oligomers prepared from partially hydrolyzed dextran were used to calibrate the elution positions as Glc units. Additional PA-tagged N-glycan standards were purchased from Seikagaku (Japan).
Sequential exoglycosidase digestions
Total or HPLC-purified PA-tagged glycans were digested with exo-glycosidases using the following conditions: -mannosidase (from jack bean, Boehringer Mannheim): 0.5 U in 100 µl of 50 mM ammonium acetate buffer containing 1 mM ZnCl2, pH 5.0; ß-galactosidase (from bovine testes, Roche): 27 mU in 100 µl of 50 mM sodium citrate phosphate buffer, pH 4.6;
-L-fucosidase (from bovine kidney, Roche): 0.1 U in 100 µl of 100 mM ammonium acetate buffer, pH 4.5;
1-3,4 fucosidase (from Xanthomonas manihotis, New England Biolabs): 5 U in 55 µl of 50 mM sodium citrate buffer, pH 6.0; ß1-4 galactosidase (from Diplococcus pneumoniae, Roche): 5 mU in 50 µl of 50 mM sodium acetate buffer, pH 6.0;
1-2,3 mannosidase (from X. manihotis, New England Biolabs): 5 U in 55 µl of 50 mM sodium citrate, pH 6.0; N-acetyl-ß-D-glucosaminidase (from D. pneumoniae, Roche): 5 mU in 50 µl of 50 mM sodium acetate buffer, pH 6.0; ß-N-acetylhexosaminidase (from jack bean, Calbiochem): 500 mU in 50 µl of 100 mM sodium citrate phosphate buffer, pH 5.0. All digestions were carried out at 37°C for 24 h. For digestion of total N-glycans, an aliquot was withdrawn from the reaction mixtures and permethylated for MS analysis of the products. The next enzyme was added without further purification. For PA-tagged samples, each digestion was additionally followed by reverse phase HPLC mapping/purification before the next digestion.
Chemical derivatization and FAB-MS analysis
Samples were permethylated using the NaOH/dimethyl sulfoxide slurry method as described by Dell et al. (1994). For FAB-MS analysis, permethyl derivatives of the N-glycans were redissolved in CH3OH for loading onto the probe tip coated with 1-monothioglycerol as matrix for positive ion modes. Alternatively, the permethylated PA-derivatives were found to run better using glycerol:m-nitrobenzylalcohol:trifluoroacetic acid (50:50:1, v/v/v) as matrix. This acidified matrix was also used in MS-MS studies to promote [M+H]+ species as the preferred parent ions. FAB-mass spectra were acquired on an Autospec orthogonal accelerationtime of flight mass spectrometer (Micromass, UK) fitted with a cesium ion gun operating at 26 kV. Collision-induced dissociation (CID) MS-MS was performed by introducing argon gas to the collision cell to a reading of
1.2 x 106 millibars on the time of flight ion gauge. The source accelerating voltage was at 8 kV with a push-out frequency of 56 kHz for orthogonal sampling. A 1-s integration time per spectrum was chosen for the time of flight analyzer with a 0.1-s interscan delay. Individual spectra were summed for data processing.
Monosaccharide composition and linkage analysis
For GC-MS linkage analysis, partially methylated alditol acetates were prepared from permethyl derivatives by hydrolysis (2 M trifluoroacetic acid, 121°C, 2 h), reduction (10 mg/ml NaBH4, 25°C, 2 h), and acetylation (acetic anhydride, 100°C, 1 h). GC-MS was carried out using a Hewlett-Packard Gas Chromatograph 6890 connected to a HP 5973 Mass Selective Detector. Sample was dissolved in hexane prior to splitless injection into a HP-5MS fused silica capillary column (30 m x 0.25 mm I.D., HP). The column head pressure was maintained at around 8.2 psi to give a constant flow rate of 1 ml/min using helium as carrier gas. Initial oven temperature was held at 60°C for 1 min, increased to 90°C in 1 min, and then to 290°C in 25 min. For monosaccharide composition analysis, released N-glycans were methanolyzed with 0.5 M methanolic-HCl (Supelco) at 80°C for 16 h; re-N-acetylated with 500 ml of methanol, 10 ml of pyridine, and 50 ml of acetic anhydride; and then treated with the Sylon HTP® trimethylsilylating reagent (Supelco) for 20 min at room temperature, dried down, and redissolved in hexane. GC-MS analysis of the trimethylsilylated derivatives was performed on the same HP system using a temperature gradient of 60°C to 140°C at 25°C/min, increased to 250°C at 5°C/min, and then increased to 300°C at 10°C/min.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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