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Glycoprotein methods protocols - biotechnology
Measurement of Sulfate in Mucins 21121117Measurement of Sulfate in MucinsMathew J. Harrison and Nicolle H. Packer1. IntroductionThe sulfation of the terminal sugar residues of mucins is a common and extensiveposttranslational event that greatly influences the ultimate viscoelastic properties ofmucin. Highly sulfated and/or sialylated mucins comprise a considerable proportionof the mucous layers of the gastrointestinal, respiratory, and reproductive tracts, andhave been demonstrated to be associated with some pathological conditions and patho-genesis (1–4). The precise biological roles for glycan sulfation are largely unknown;however, several groups have demonstrated discrete biological roles for specificinstances of glycan sulfation of non-mucin glycoproteins. These roles include the con-trol of the circulatory half-life of human luteinizing hormone, symbiotic interactionsof leguminous plants and nitrogen-fixing bacteria, and the targeting of lymphocytes tolymph nodes (5).Historically, efforts to assess qualitatively and quantitatively the extent of glyco-protein sulfation have been based on the metabolic incorporation of 35S. However, thismethod is expensive and painstaking to perform. Sulfate and phosphate are currentlyindistinguishable by normal modes of mass spectrometric analysis (79.96 and 79.97Dalton, respectively), although collision-induced fragmentation electrospray massspectrometry (MS) has been used to distinguish between phosphate and sulfate on afungal glycoprotein-cellobiohydrolase I (6). Others have also used tandem MS to as-sign sugar-sulfate linkages on rat MUC2 (7).Here we present a chromatographic method for the identification and quantitationof sulfation on mucin. This method is comparatively cheap and fast to perform, andcan be automated to provide fast-screen or high-throughput analysis. It is particularlyeasy to implement for groups who are currently performing mucin sugar analysis,since much of the necessary chromatographic equipment and the hydrolysis methodare similar for both methods. Although this method is not particularly sensitive bytoday’s analytical standards ( >1 nmol), it provides fast and reproducible quantitationof sulfate for highly sulfated biomolecules such as mucins.From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 212 Harrison and Packer2. Materials2.1. Solvents and Reagents1. Water, carbonate-free (see Note 1), and sparged with a constant flow of argon.2. 0.1 M NaOH, made up with decarbonated water, and similarly sparged with argon.3. At least 2 L of 50 mM sulfuric acid (see Note 2).4. HCl, concentrated (Aristar, BDH Laboratory Supplies, Poole, Dorset, UK).5. 1 mM 2-sulfo-N-acetylglucosamine, 3-sulfo-N-acetylglucosamine, and 6-sulfo-N-acetylglucosamine, in water (see Note 3).2.2. Equipment1. High-performance liquid chromatography (gradient controller, binary pump) (see Note 4).2. AS11 (4 × 250 mm) ion-exchange column (Dionex) (see Note 5).3. AMMS II postcolumn ion suppressor (Dionex, Corp., Sunnyvale, CA).4. Conductivity detector (Dionex).5. Heating block, set to 100°C, or boiling water bath.6. Vacuum centrifuge (Savant Speedvac™, Savant Instruments, Farmingdale, NY).3. Methods3.1. Sample Hydrolysis1. Transfer an appropriate volume (see Note 6) of each desalted sample (see Note 7) to a separatescrew-top (or otherwise tightly sealable) Eppendorf tube, and dry completely in a Speedvac.2. Resuspend each sample in 50 µL of 4 M HCl and mix thoroughly by vortexing.3. Transfer to a 100°C heating block and hydrolyze for precisely 4 h.4. After briefly centrifuging to spin condensate to the bottom of the tube, transfer the hydro-lyzed samples to a vacuum centrifuge and dry completely. When dry, add 50 µL of waterto each sample and dry again, in order to reduce the levels of chloride (expunged asgaseous HCl).3.2. Analysis of Sulfate by Ion Chromatography3.2.1. ChromatographyFigure 1 presents typical elution positions of ionic species using this method.1. Attach the AS11 ion column to the high-performance liquid chromatograph. Attach theion suppressor downstream of the column, and the conductivity cell downstream of theion suppressor, according to the manufacturer’s recommendations. Attach the neutraliz-ing counterflow (50 mM sulfuric acid) reservoir to the ion suppressor.2. The initial chromatographic conditions are 5 mM NaOH (95% water, 5% 0.1 M NaOH) ata flow rate of 1 mL/min. Sulfuric acid is used as the neutralizing countercurrent in the ionsuppressor at a flow rate of about 3 mL/min (see Note 8).3. Resuspend dry, hydrolyzed samples in an appropriate (25–100 µL) volume of water. Atypical injection volume is 25 µL.4. Perform chromatography over the following curved gradient:a. 0 to 10 min: injection at t = 0, curved gradient (curve = 8) from 5 mM NaOH (95%water, 5% 0.1 M NaOH) to 30 mM NaOH (70% water, 30% 0.1 M NaOH).b. 10 to 11 min: isocratic gradient (curve = 5) at 30 mM NaOH.c. 11 to 15 min: curved gradient (curve = 1) from 30 mM NaOH to 5 mM NaOH.d. 16 to 25 min: re-equilibration in 5 mM NaOH. Measurement of Sulfate in Mucins 2133.2.2. Quantitation of SulfateReleased inorganic sulfate can be quantitated by comparison to an external control,consisting of monosaccharide sulfates that are hydrolyzed at the same time (see Note 9).1. Take 3 and 15 (total) nmol of a mixture of monosaccharide sulfates and hydrolyze as inSubheading 3.1.2. Calculate the amount of sulfate from the area of the sulfate peak as follows:No. nmol sulfate = (Areamucin sulfate peak – Area–ve control sulfate peak)/Area1 nmol sulfate3. Figure 2 shows a sample analysis of human MUC2 glycopeptides A and B (kindly sup-plied by I. Carlstedt, University of Lund, Sweden). An average value of 79 nmoles ofsulfate from 50 mg of mucin glycopeptides was established (see Note 10).4. Notes1. Carbonate ions (CO32-) elute at a retention time close to that of sulfate. It is necessary todecarbonate water by boiling in a 2-L conical flask covered with an upturned beaker.After 5–10 min of rapid boiling, transfer the hot flask to an ice bath or similar, andsparge the decarbonated water with a constant stream of argon until the water is approx-imately at room temperature. Make up the hydroxide eluent, taking care to minimizeexposure to air.2. Large volumes of 50 mM sulfuric acid are consumed in the neutralization of thepostcolumn flow by the ion suppressor before analytes can be detected by the conductiv-ity cell. Allow approx 250 mL/h of chromatography.3. Note that carbohydrates are hygroscopic and must be thoroughly dried before weighingand making up as standards. Standards of 1 mM each of 2-, 3-, and 6-sulfo-N-acetylglucosamine in water may be aliquoted and stored at –20°C, and used as required.Fig. 1. Elution positions of acetate (CH3COO–), chloride (Cl–), sulfate (HSO4–), and phos-phate (H2PO3–) ions (see also Note 11). Chromatogram displays 10 nmol each of phosphateand sulfate, and trace amounts of chloride and acetate. Note the difference in detector responsebetween equivalent amounts of sulfate and phosphate. 214 Harrison and Packer4. Our system for sulfate analysis uses the same chromatographic system as that for sugaranalysis, in which the ion chromatography column and high-performance anion-exchangechromatography (HPAEC) column are connected to a conductivity cell and pulsedamperometric detector, respectively. Since both modes of chromatography use the samesolvents, either ion chromatography or monosaccharide-HPAEC is achieved by simplyswitching the column inlet to the desired column and selecting the appropriate detector.5. The Dionex AS11 ion-exchange column was originally marketed as a strong anion-exchange (Omni-Pac™ PAX-100) column and was used extensively for the analysis ofinorganic ions.6. The limit of detection of sulfate by ion chromatography as described here is about 1 nmol.Since mucins are often highly sulfated (i.e., the number of moles of sulfate >> the numberof moles of protein), the amount of protein required for analysis will often be quite small.For example, 10 pmol of a highly sulfated mucin (>100 mol sulfate/mol of protein) wouldbe adequate for a confident analysis, whereas the sulfation of a protein that contains aunique sulfation site (1 mol sulfate/mol of protein) would not be detectable with <1 nmolof protein. Because the molecular weight of mucins is difficult to determine, the molaramount of the mucin is usually unknown. However, if we generalize and estimate anaverage molecular weight of 1 MDalton, then 10–100 µg of mucin should be hydrolyzedin the first instance.7. Sample preparation and handling prior to hydrolysis are important factors affecting theaccuracy and precision of sulfate analysis. Naturally, samples must be free from buffersthat contain phosphate and/or sulfate (i.e., no phosphate-buffered saline!), as well as sul-fate-phosphate-containing detergents (e.g., sodium dodecyl sulfate), chaotropes (e.g.,thiourea), and reducing agents (e.g., mercaptoethanol). Ideally, desalting into water shouldbe performed as a matter of course prior to sulfate analysis. For proteins that have poorsolubility in water, consider desalting into partially organic solvents such as 50% aceto-nitrile or 50% isopropanol, or volatile buffers such as ammonium acetate or ammoniumbicarbonate. If desalting is not possible or cannot reliably be performed, sulfate may bedetermined subtractively by direct injection of a known volume of unhydrolyzed samplein order to determine the amount of sulfate present as free (unbound) sulfate. This valueis then subtracted from the value obtained after hydrolysis of an equivalent volume.Fig. 2. Quantitation of sulfation of glycopeptides A and B from human MUC2. The chro-matogram shown corresponds to half of an original hydrolysis amount of 50 µg. Measurement of Sulfate in Mucins 2158. A flow rate of approx 3 mL/min of 50 mM sulfuric acid is sufficient to produce a stable baselineover the course of the separation method. Note that the flow rate of sulfuric acid to the ionsuppressor does not need to be precise; in our laboratory the flow of sulfuric acid to the suppres-sor is regulated by positive pressure from an air cylinder. Upward baseline drift is diagnostic ofan insufficient flow rate of sulfuric acid to the ion suppressor. However, an excessive flow rateof sulfuric acid to the suppressor is not responsible for downward baseline drift.9. To quantitate sulfate, it is necessary to calibrate the analysis to an external control that ishydrolyzed under the same conditions. Adding an inorganic ion as an internal standardrisks inaccuracies from ionic contaminants. Since there are differences in sulfate yieldfrom sugar sulfates of different linkage, even after 4 h of hydrolysis (linkage lability: 6- >2- > 3-), we recommend including two external controls that contain an equimolar mix-ture of 2-, 3- and 6-sulfo-N-acetylglucosamine in water: a 3 nmol control (1 nmol of eachsugar sulfate), and a 15 nmol control (5 nmol of each sugar sulfate). Alternatively, anadequate quantitation can be obtained by using an external control of hydrolyzed inor-ganic Na2SO4. The use of only inorganic sulfate as a calibrant will give a slightly lowersulfate quantitation than the use of monosaccharide sulfates. Note that it is also necessaryto include a negative control, in which a volume of water is hydrolyzed under the sameconditions, and any reagent sulfate contamination can be subtracted.10. Since the molecular weight of mucins is difficult to determine, the number of moles ofsulfate may be expressed either as per nanomole of total amino acids, as determined byamino acid analysis (see Chapter 10), or as per nanomoles of total monosaccharides, asdetermined by HPAEC-pulsed amperometric detection.11. Note that in this chromatographic method, monosaccharide-6-phosphates elute with simi-lar retention times to those of inorganic sulfate, although under the hydrolysis conditionsdescribed, monosaccharide phosphates quantitatively release to phosphate. Note also thatinorganic phosphate is clearly resolved by this chromatography and can also be quantitated.The hydrolysis of MUC2 glycopeptides (Fig. 2) showed the presence of inorganic phos-phate, which was not present in the unhydrolyzed sample. Monosaccharide-sulfates havenot been observed to elute from the column under the method described here.AcknowledgmentsThis work was supported by an Australian Proteome Analysis Facility IndustryResearch Development scholarship, Macquarie University. The authors acknowledgesthe support of the National Health and Medical Research Council, Australia.References1. Corfield, A. P., Myerscough, N., Bradfield, N., Corfield, C. D. A., Gough, M., Clamp, J. R.,Durdey, P., Warren, B. F., Bartolo, D. C. C., King, K. R., and Williams, J. M. (1996) Colonicmucins in ulcerative colitis: Evidence for loss of sulfation. Glycoconj. J. 13, 809–822.2. Ishikawa, N., Shi, B. B., Khan, A. I., and Nawa, Y. (1995) Reserpine-induced sulphomucinproduction by goblet cells in the jejunum of rats and its significance in the establishmentof intestinal helminths. Parasite Immunol. 17, 581–586.3. Lo-Guidice, J. M., Herz, H., Lamblin, G., Plancke, Y., Roussel, P., and Lhermitte, M. (1997)Structures of sulfated oligosaccharides isolated from the respiratory mucins of a non-secre-tor (O, Le-a+b-) patient suffering from chronic bronchitis. Glycoconj. J. 14, 113–125.4. Veerman, E. C. I., Bank, C. M. C., Namavar, F., Appelmelk, B. J., Bolscher, J. G. M., andNieuw Amerongen, A. V. (1997) Sulfated glycans on oral mucin as receptors forHelicobacter pylori. Glycobiology 7, 737–743. 216 Harrison and Packer5. Hooper, L. V., Manzella, S. M., and Baenziger, J. U. (1996) From legumes to leukocytes:biological roles for sulfated carbohydrates. FASEB J. 10, 1137–1146.6. Harrison, M. J., Nouwens, A. S., Jardine, D., Zachara, N. E., Gooley, A. A., Nevalainen,H., and Packer, N. H. Eur. J. Biochem. 256, 119–127.7. Karlsson, N. G., Johansson, M. E., Asker, N., Karlsson, H., Gendler, S. J., Carlstedt, I., andHansson, G. C. (1996) Molecular characterization of the large heavily glycosylated domainglycopeptide from the rat small intestinal MUC2 mucin. Glycoconj. J. 13, 823–831. . UK).5. 1 mM 2-sulfo-N-acetylglucosamine, 3-sulfo-N-acetylglucosamine, and 6-sulfo-N-acetylglucosamine, in water (see Note 3).2.2. Equipment1. High-performance. (linkage lability: 6- > 2- > 3-) , we recommend including two external controls that contain an equimolar mix-ture of 2-, 3- and 6-sulfo-N-acetylglucosamine