Saliva is a specialized, dilute, aqueous secretion produced by three major exocrine glands-the parotid, submandibular and sublingual-and several minor glands in the mouth. Functions of saliva include lubrication of the oral cavity and non-immune defense against pathogens in the environment (Wu et al., 1994; Veerman et al., 1995). Human saliva contains several components implicated in the protection of the oral cavity, including secretory IgA, lysozyme, proline-rich proteins, histatins, and mucins (Iontcheva et al., 1997). These latter macromolecules are large glycoproteins with O-linked glycans, and human saliva has been shown to contain at least two structurally and functionally distinct populations of mucins which are divided into the high molecular weight (Mr > 106 Da) oligomeric, gel-forming MG1 population and the lower molecular weight (Mr 1.2-1.5 × 105 Da) monomeric MG2 mucins (Loomis et al., 1987; Ramasubba et al., 1991; Veerman et al., 1992). The polypeptide chain of the MG2 population has been identified as the product of the MUC7 gene (Bobek et al., 1993). The MG1 population is heterogeneous (Thornton et al., 1995) and it has been demonstrated that the different salivary glands secrete distinct populations of MG1, which vary in buoyant density (Veerman et al., 1992) and the degree of sulfation and sialylation of their glycan chains (Bolscher et al., 1995). cDNA cloning studies and Northern blotting data indicate that the MUC5B gene product forms a major constituent of MG1 (Troxler et al., 1995, 1997; Nielsen et al., 1997). However, at present it is unclear whether one or a number of gene products constitute the MG1 mucins.
In this study we have attempted to identify the polypeptide(s) underlying the MG1 population of mucins. We have demonstrated the presence of two distinct populations of MG1 mucins, which vary in their carbohydrate content and their charge density. Amino acid compositional analysis of the glycopeptides and MALDI-TOF MS analysis of tryptic peptides from both populations indicate they are differently glycosylated products of the MUC5B gene, and therefore we propose that the MUC5B gene product is the predominant mucin species of MG1.
Mucin purification
Saliva contains two families of mucin termed MG1 and MG2 (MUC7) which can be distinguished on the basis of their molecular size. Gel chromatography on Sepharose CL-4B (data not shown) separated the high-Mr mucins, corresponding to MG1, which were eluted in the void volume of the column from the smaller MG2 mucins which were more included (Mehrotra et al., 1998). The MG1-containing fractions were further purified by density-gradient centrifugation, first in CsCl/4 M guanidinium chloride to remove proteins and then in CsCl/0.2 M guanidinium chloride to remove nucleic acids. Typical profiles from such a purification are shown in Figure
Figure 1. Density gradient centrifugation of MG1 mucins from Sepharose CL-4B in (a) CsCl/ 4M guanidinium chloride and (b) CsCl/ 0.2M guanidinium chloride. (a) Density-gradient centrifugation in CsCl/ 4M guanidinium chloride was performed as described in the experimental section. Tubes were emptied from the top and fractions (5 ml) were assayed for carbohydrate with the PAS assay (solid line), nucleic acids and proteins by absorbance measurements at 280 nm (broken line) and the density determined by using a Hamilton syringe as a pycnometer. Mucin-containing fractions were pooled as indicated prior to (b) centrifugation in CsCl/0.2 M guanidinium chloride. Tubes were emptied and assayed as above. Mucin containing fractions were pooled as indicated with the horizontal bars. Reduced MG1 mucin subunit fractionation
Reduced MG1 mucin subunit preparations from three individuals were fractionated by analytical anion-exchange chromatography and the carbohydrate-profiles, as monitored with a PAS-assay, are presented in Figure
Figure 2. Anion-exchange chromatography of reduced MG1 mucin subunit preparations. Reduced mucin subunits from three individuals (a-c) were chromatographed on a Mono Q HR5/5 column as described in Materials and methods. Fractions (0.5 ml) were assayed after immobilization onto nitrocellulose with the PAS-reagent (solid line) and the nominal elution gradient is shown (dashed line).
Table I. Reduced MG1 mucin carbohydrate analysis
The neutral sugar compositions of the reduced MG1 subunits in the fractions across the anion exchange distribution were determined and a graphical representation of the data (Figure
Figure 3. Analysis of the reduced MG1 mucin subunits after anion-exchange separation. The data presented are on the anion exchange distribution shown inFigure 2a. Aliquots of fractions were taken for determination of (a) their total neutral sugar composition and their reactivity with anti-carbohydrate monoclonal antibodies (b) INES and (c) F2. In (b) the membrane was treated with neuraminidase prior to probing with the antibody. In addition the contents of (d) the monosaccharides Fuc (black bars), GlcNAc (white bars) and Gal (gray bars) normalized to GalNAc and (e) the percentage of the amino acids Ser (black bars), Thr (white bars), and Pro (gray bars) are shown for the mucin subunit-containing fractions. (f) Fractions were also analyzed for their reactivity with the MUC5B-mucin specific antiserum (MAN-5B-I). The nominal elution gradient is shown (dashed line).
Table II. Reduced MG1 mucin polypeptide identification
The amino acid composition of selected fractions from the anion exchange column was determined and the serine, threonine and proline contents of the MG1 subunits are shown in Figure
The antibody data above suggests MUC5B is a major mucin in the MG1 preparation; however, it does not tell us whether it is the only mucin present. To ascertain what proportion of the MG1 preparation was accounted for by the MUC5B mucin, tryptic mapping and further amino acid analyses were performed. Prior to this it was necessary to prepare larger quantities of reduced MG1 subunits and this was accomplished by preparative anion exchange chromatography (data not shown). Fractions from the column were pooled to yield two groups of molecules corresponding to the two differently charged mucin populations A and B, and these were subjected to trypsin treatment. The degradation products gave similar elution profiles after gel chromatography on Superose 12 (Figure
Mono Q fraction
Mr (×10-6 Da)
RG (nm)
28
2.8
53
30
2.5
53
32
2.6
56
34
2.8
58
36
2.8
59
38
2.6
60
40
2.9
64
42
2.6
61
44
2.5
59
Monosaccharide
Mucin populationa
A
B
A
B
(nmol/5 µg mucin)
(normalized to GalNAc)
Fuc
4.1
1.1
2.6
1.8
GalNAc
1.6
0.6
1.0
1.0
GlcNAc
4.7
1.5
2.9
2.5
Gal
6.8
1.9
4.2
3.2
Man
2.8
2.8
1.8
4.7
Total neutral
19.9
7.9
Sugars
Sialic acid
0.3
0.15
0.2
0.25
Sulfate
0.05
0.12
Table III.
High Mr-mucin glycopeptide fraction | |||
Amino acid | A-I | B-I | MUC5B mucin glycopeptidea |
Asx | 10 | 13 | 10 |
Glx | 34 | 34 | 34 |
Ser | 178 | 168 | 172 |
Gly | 64 | 64 | 56 |
Thr | 290 | 295 | 290 |
Ala | 98 | 103 | 129 |
Pro | 187 | 169 | 180 |
Arg | 18 | 19 | 19 |
Tyr | 5 | 5 | 4 |
Val | 38 | 40 | 35 |
Ileu | 20 | 20 | 16 |
Leu | 46 | 45 | 39 |
Phe | nd | 7 | 14 |
Lys | 12 | 17 | 2 |
Table IV.
A-II | B-II | MUC5B mucina |
1032 | 1035 | 1038 |
1128 | 1132 | 1129 |
1683 | 1688 | 1685 |
1976 | 1978 | 1975 |
Table V.
Gland | Mr (× 10-6 Da) | RG (nm) |
Palatal | 19 | 222 |
Sublingual | 32 | 271 |
Submandibular | 20 | 191 |
Whole saliva | 25 | 230 |
Mucins produced by individual salivary glands
Mucins, purified as above from whole saliva and from the secretions isolated from sublingual, submandibular, and palatal glands, were reduced and alkylated, and the resulting reduced subunits were subjected to anion exchange chromatography (Figure
The average molecular weight and size of the intact mucins from whole saliva and the three glands was determined by in-line laser light scattering after chromatography on Sephacryl S-1000 (data not shown) and the values are presented in Table V. The MUC5B mucins purified from whole saliva had Mr in the range 14-40 × 106 Da with an average Mr of 25 × 106 Da. The mucins from the palatal and submandibular glands were lower in molecular weight than the total population whereas the sublingual gland mucins were larger. The size distribution of the MUC5B mucins produced by each of the salivary glands was assessed by rate zonal centrifugation (Figure
In the present study we have isolated the high-Mr MG1 salivary mucins by exploiting their large hydrodynamic volume (void volume CL-4B) and high buoyant density (1.35-1.55 g/ml in 4 M guanidinium chloride/CsCl and 1.40-1.52 g/ml in 0.2 M guanidinium chloride/CsCl). In common with the gel-forming mucins isolated from both respiratory tracts (Thornton et al., 1990) and cervical tracts (Carlstedt et al., 1983), the mucins are oligomeric and can be fragmented into their constituent subunits (Mr 2.5-2.9 × 106) by reduction of disulfide bonds. The MG1 mucin preparations contain two populations of molecules that differ with respect to their charge density. This is in agreement with previous studies which have shown that the high molecular weight MG1 mucins are heterogeneous and that the major salivary glands secrete differently charged forms of these mucins (Veerman et al., 1991, 1992; Bolscher et al., 1995). The identity of the polypeptides underlying these mucins has not previously been elucidated. Recent cDNA cloning data indicates that the MUC5B mucin is present in the MG1 population (Nielsen et al., 1997; Troxler et al., 1997), but its proportion is undetermined. Using a combination of antibody reactivity, peptide mapping and amino acid analysis, we have demonstrated that the polypeptide of the majority of the mucins in the MG1 preparation are encoded for by the MUC5B gene. While it may be argued that antibody reactivity alone cannot give quantitative information on the relative abundance of the MUC5B mucin, this is not so for the two chemical analyses. Both peptide mapping and amino acid analysis indicate that MUC5B is the predominant mucin, and we are unable to find evidence for significant quantities of other mucins in the MG1 preparation. There is a remarkable similarity in the amino acid composition between the MG1 high-Mr glycopeptides and those derived from the MUC5B mucin from the respiratory tract (Thornton et al., 1997). Furthermore, the mass fingerprints of the peptides derived from the MG1 mucins are very similar to those derived from the respiratory MUC5B mucins (Thornton et al., 1997). The peptides identified in the previous study were found to be present within a contiguous 51 amino acid sequence, and we proposed on the basis of their abundance that the sequence would be repeated numerous times throughout the mucin polypeptide. Indeed, recent cDNA cloning data has confirmed the presence of this repetitive motif within the MUC5B mucin polypeptide (Desseyn et al., 1997).
Figure 4. Agarose gel electrophoresis of the fractionated reduced MG1 mucin subunits. Aliquots of selected fractions from the Mono Q separation of the reduced mucin subunits were electrophoresed on a 1% (w/v) agarose gel and then Western blotted onto nitrocellulose as described in Materials and methods. The blot was probed (a) with the MUC5B-mucin specific antiserum (MAN-5B-I) and then stripped and reprobed (b) with the general mucin subunit antiserum (MAN-SUBS).
Figure 5. Superose 12 chromatography of trypsin digestion products of reduced MG1 mucin subunit populations A and B. The reduced mucin subunit populations (a) A and (b) B were treated with trypsin and subsequently chromatographed on a Superose 12 HR 10/30 column. The column eluent was monitored for absorbance at 280 nm (broken line) and with the PAS-reagent (solid line). Fractions from the column runs were pooled as follows: A-I 6-12, A-II 13-22, B-I 6-12, and B-II 13-22. These poolings are indicated by the bars.
Recently, mucin gene expression has been studied in the sublingual and submandibular glands by Northern blot analyses (Troxler et al., 1997). The data indicated that MUC5B is the major known mucin expressed in these tissues. MUC4 RNA was shown to be expressed weakly by the two glands, but MUC2, MUC3, MUC5AC, and MUC6 expression were not detected. As far as the MUC2 and MUC5AC mucins are concerned, our antibody data are consistent with these observations. On the basis of the biosynthetic capacity of the submandibular glands, Troxler et al. (1997) suggest that other as yet unidentified mucins are secreted from these glands and contribute to the MG1 population. Our data suggests that if other mucins are secreted they do not form a significant part of the MG1 preparation studied here. However, we do not rule out that either smaller mucins or mucins with a different density are produced from the submandibular glands, which would be excluded from our preparation.
The MUC5B reduced mucin subunits are heterogeneous in their charge distribution and two major populations of molecules can be observed. We have previously noted differently glycosylated populations of this mucin in respiratory tract secretions (Thornton et al., 1997). The higher charge population in saliva appears to originate from the palatal glands, and the proportion of the MUC5B mucins in whole salivas contributed to by the palatal glands can vary. It would appear that the palatal gland MUC5B mucin is a minor component compared to the mucins from the sublingual and submandibular glands, which are of similar but lower charge density. The two populations of MUC5B mucins can be distinguished on the basis of glycan-epitope specific monoclonal antibody, and there is evidence that the lower charged form has a higher fucose content. However, carbohydrate compositional data indicates the two populations may have similar glycan structures.
The heterogeneity in charge density of the MUC5B mucin subunits is not accompanied by any significant change in their molecular weights. Furthermore, the sialic acid content of the mucins is similar. However, the high charged subunit population B has a 2- to 3-fold increase in sulfate content compared to the less acidic population A. Thus, the increasing acidity observed by anion exchange chromatography and the increasing electrophoretic mobility in agarose gels of the mucin subunits must be due to their increasing levels of sulfation. The sulfate is probably present as SO3-3Gal[beta]1-3GlcNAc moieties since the more charged molecules are reactive with the monoclonal antibody F2 which recognizes this portion of the sulfo-Lewisa antigen. (Veerman et al., 1997).
The MUC5B mucins isolated from whole saliva and the different salivary glands are polydisperse in size. This polydispersity must arise from a variable number of subunits in the intact oligomeric mucins. Thus, for example, the mucins in whole saliva (Mr 14-40 × 106) are constructed from between 5-16 subunits (Mr 2.5-2.9 × 106) and these multi-subunit assemblies are stabilized by disulfide bonds. The MUC5B mucins from sublingual gland are of highest average molecular weight, whereas those from the palatal and submandibular glands are on average smaller. It is interesting to note that the MUC5B mucins in the palatal secretions have the broadest spread of molecular sizes, with a proportion of molecules possibly as small as monomers. Conclusions
The MUC5B mucin is the predominant mucin in the salivary MG1 preparation and is found in different glycosylated forms. The mucins differ in their relative levels of sulfation and fucose, the most charged (most sulfated) form of the mucin arises from the palatal glands. The intact MUC5B mucins are on average very large; however, there is evidence that the palatal gland mucins contain a significant proportion of smaller species.
Figure 6. MALDI-TOF mass spectrometry of the low-Mr tryptic peptides derived from reduced mucin subunit populations A and B. Tryptic peptide-containing fractions (a) A-II and (b) B-II were analyzed by MALDI-TOF MS. The peak heights represent the relative abundance ofeach species. Highlighted with asterisks are the major peptides isolated and sequenced after trypsin digestion of the MUC5B mucin isolated from respiratory mucus (Thornton et al., 1997).
Figure 7. Anion-exchange chromatography of the reduced mucin subunit preparations from individual salivary glands. Mucins isolated from (a) whole saliva and (b) palatal, (c) sublingual, and (d) submandibular glands, were reduced and alkylated and the resulting reduced mucin subunits chromatographed on a Mono Q HR5/5 column as described in Materials and methods. Fractions (0.5 ml) were assayed after immobilization onto nitrocellulose with the PAS-reagent (solid line) and for reactivity with the MUC5B-mucin specific (MAN-5B-I) antiserum (open circles). The nominal elution gradient is shown (dashed line).
Agarose UltraPURE (electrophoresis grade) was from BRL (Paisley, UK). The ECL Western detection kit was from Amersham International Plc. (Amersham, UK). Neuraminidase (EC 3.2.1.18; type X from Clostridium perfringens), Tween 20, (3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propane-sulfonate) or CHAPS, Schiff's reagent, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, [alpha]-cyano-4-hydroxy cinnamic acid, Substance P, insulin (bovine pancreas) and guanidinium chloride (practical grade) were purchased from Sigma Chemical Co. (Poole, UK). Stock solutions of guanidinium chloride (~8 M) were treated with charcoal before use. Trypsin, modified by reductive alkylation to reduce autolysis, was purchased from Promega (Southampton, UK). Saliva collection
Glandular secretions were collected separately using custom fitted devices as described previously (Veerman et al., 1996). Secretions were collected in ice-cooled vessels containing 8 M GuHCl and a cocktail of protease inhibitors (final concentrations 10 mM EDTA, 5 mM benzamidine hydrochloride, and 100 mM [epsis]-aminocaproic acid). Whole saliva was stimulated by chewing on parafilm and collected directly into the extraction buffer. Preparation of mucins
Mucins were extracted from saliva (2 days, gentle stirring, 4°C) with 6 M guanidinium chloride/10 mM sodium phosphate buffer/0.05% (w/v) CHAPS, pH 6.5 containing proteinase inhibitors (10 mM EDTA/10 mM N-ethylmaleimide/100 mM [epsis]-amino-n-caproic acid/ 5mM benzamidine hydrochloride) and purified by Sepharose CL-2B chromatography and density-gradient centrifugation. Briefly, the extract was spun at 4400 × g for 10 min at 4°C and the supernatant chromatographed on a column of Sepharose CL-4B (48 cm × 5cm) eluted with 4 M guanidinium chloride/10 mM sodium phosphate buffer/0.1% (w/v) CHAPS, pH 6.5, at a flow rate of 40 ml/h. The mucins eluted in the void volume of the column (MG1) were then subjected to CsCl/4 M guanidinium chloride density-gradient centrifugation at a starting density of 1.4 g/ml in a Beckman Ti45 angle rotor at 40,000 r.p.m. for 67 h at 15°C. The mucin (MG1) containing fractions were pooled as shown in Figure
Figure 8. Rate-zonal centrifugation of the MUC5B mucins isolated from different salivary glands. Mucins purified from (a) palatal, (b) sublingual, and (c) submandibular gland secretions were centrifuged on 6-8 M guanidinium chloride gradients as described in Materials and methods. Gradients were emptied from the top into 0.5 ml fractions and aliquots blotted onto nitrocellulose and analyzed for carbohydrate with a PAS assay. Preparation of reduced mucin subunits
Reduced mucin subunits were obtained by treatment of the purified mucins in 6 M guanidinium chloride/0.1 M Tris, pH 8.0, with 10 mM dithiothreitol for 5 h at 37°C. Iodoacetamide was then added to a final concentration of 25 mM, and the mixture was left in the dark overnight at room temperature. Reduced mucin subunits were dialyzed into 6 M guanidinium chloride and stored at 4°C. Anion-exchange chromatography
Reduced mucins were transferred from 6M guanidinium chloride into 6 M urea/10 mM piperazine pH 5.0 containing 0.02% CHAPS by chromatography on a Pharmacia PD-10 column prior to separation by anion-exchange chromatography. Reduced mucins were then chromatographed on a Pharmacia Mono Q HR 5/5 column eluted with a linear gradient of 0-0.4 M lithium perchlorate/10 mM piperazine pH 5.0 in 6M urea containing 0.02% CHAPS (Thornton et al., 1995). Agarose gel electrophoresis
Reduced mucin subunits were electrophoresed in 1% (w/v) agarose gels in 40 mM Tris-acetate/1 mM EDTA pH 8.0 containing 0.1% (w/v) SDS, for 30 V and 18 h at room temperature. After electrophoresis, subunits were transferred to nitrocellulose by vacuum blotting prior to detection of mucins using antibodies (Thornton et al., 1995). Bands were visualized using horseradish peroxidase-labeled secondary antibodies in conjunction with an ECL Western detection kit. Antibodies
As a general nonspecific mucin probe, a polyclonal antiserum raised against cervical mucin reduced subunits (MAN-SUBS) that recognizes protein epitopes on a range of reduced mucins was employed (Sheehan et al., 1991). Mucin-specific polyclonal antisera that recognize the reduced subunits of the MUC2 (Sheehan et al., 1996), MUC5AC (Thornton et al., 1996), and MUC5B mucins (Thornton et al., 1997) were also used. Monoclonal antibody F2 recognizes the SO3-3Gal[beta]1-3GlcNAc moiety of the sulfo-Lewisa antigen. (Veerman et al., 1997). Monoclonal antibody INES was elicited against a purified high-Mr salivary mucin species (Rathman et al., 1990). This monoclonal antibody recognizes a periodate sensitive epitope on salivary mucins and removal of sialic acid enhances its binding to antigen. Rate-zonal centrifugation
Rate zonal centrifugation was performed essentially as described previously (Thornton et al., 1990). In brief mucins were loaded onto preformed 6-8 M guanidinium chloride gradients and centrifuged in a Beckman SW 40Ti swing out rotor at 40,000 r.p.m. for 1.5 h at 15°C. Light scattering
Intact mucins were chromatographed on a Sephacryl S-1000 column, and reduced mucin subunits were chromatographed on a Pharmacia Superose 6 HR 10/30 column. Both columns were eluted with phosphate-buffered saline, pH 7.4, at a flow rate of 0.5 ml/min. The column eluate were passed through an in-line Dawn DSP laser photometer and a Wyatt/Optilab 903 interferometric refractometer (Optichem, Clywd, UK) to measure light scattering and sample concentration, respectively. Light scattering measurements were taken continuously at 18 angles between 15° and 151° and the data analyzed according to Zimm (1947). Preparation of high-Mr glycopeptides and tryptic peptides
Reduced mucin subunits were dissolved in 0.1 M ammonium hydrogen carbonate, pH 8.0, and digested with trypsin for 24 h at 37°C. The digest was chromatographed on a Superose 12 HR 10/30 column in 0.1 M ammonium hydrogen carbonate, pH 8.0, at a flow rate of 0.4 ml/min and fractions from the column were combined into two pools (Figure MALDI-TOF mass spectrometry
Samples (1 µl) in 0.1% TFA were mixed with an equal volume of 50 mM [alpha]-cyano-4-hydroxy cinnamic acid, applied to a TOFspec target and analyzed by MALDI-TOF MS in linear positive ion mode using a VG TOFSpec-E with Substance-P (M+ 1348.7 Da) and bovine insulin (M+ 5734.5 Da) as internal standards. The data generated were processed using the OPUS peak detection program. Amino acid analysis of high-Mr mucin glycopeptides
Samples were hydrolyzed under nitrogen in 3M HCl at 105°C for 16 h, and the resulting amino acids were derivatized with phenyl isothiocyanate and then separated by reverse-phase chromatography using a 3 µm ODS2 column. Compositional analysis of MG1 reduced mucin subunits separated by anion exchange chromatography
Reduced MG1 mucin species separated by Mono Q anion exchange chromatography were immobilized on PVDF membrane and were hydrolyzed and analyzed by HPAEC-PAD for monosaccharide composition as described by Packer et al. (1996). Amino acid composition of PVDF blotted mucin fractions was determined by pre-column FMOC derivatization essentially as in Yan et al. (1996) except that the mucins were acid hydrolyzed in 6N HCl at 105°C for 24 h to prevent charring. Sulfate analysis
Samples (20 µg) were hydrolyzed in 4 M HCl for 4 h at 100°C. The samples were dried in a Speed-Vac concentrator and washed once with water. The sulfate was separated from other ions by HPLC on an AS11 (4 × 250 mm) ion-exchange column (Dionex) using an AMMS II post-column ion suppressor (Dionex) with conductivity detection. A gradient of 5 mM to 30 mM NaOH over 10 min, followed by 5 min at 30 mM NaOH, at a flow rate of 1 ml/min was used to elute the anions. Sulfuric acid (50 mM) was used as the neutralizing counter-current in the ion suppressor at a flow rate of about 3 ml/min. Quantitation was by external calibration with 1 nmol sodium sulfate. Analytical methods
Slot blotting of chromatographic fractions and PAS staining was carried out as previously described (Thornton et al., 1989). Antibody detection of slot blotted fractions was as described by Thornton et al. (1994). Blots were visualized using horse radish peroxidase-labeled secondary antibodies in conjunction with an ECL Western detection kit or alkaline phosphatase-labeled secondary antibodies using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as the chromogenic substrate. Color yields were quantitated essentially by reflectance densitometry as described by Thornton et al. (1989).
N.H.P. thanks Margaret Lawson for excellent technical assistance. D.J.T. and J.K.S. thank the Wellcome Trust for financial support. N.H.P. thanks the National Health and Medical Research Council of Australia for financial support.
MG1, high-Mr salivary mucins; MG2, low-Mr salivary mucins; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PAS, periodate Schiff's; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine, Fuc, fucose; Gal, galactose; Man, mannose; ECL, enhanced chemiluminescence; HPAEC-PAD, high-pH anion exchange chromatography with pulsed amperometric detection. CHAPS, (3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propane-sulfonate).
3To whom correspondence should be addressed
Materials and methods
Acknowledgments
Abbreviations
References
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