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Glycoprotein methods protocols - biotechnology
Amino Acid Analysis of Mucins 113113From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ10Amino Acid Analysis of MucinsJun X. Yan and Nicolle H. Packer1. IntroductionAmino acid analysis is a commonly used technique that provides quantitative esti-mation of the amounts of proteins/amino acids present in a sample and/or qualitativeinformation on the amino acid composition of a protein. For protein analysis, the tech-nique essentially involves acid hydrolysis of amino acid peptide bonds within the pro-tein; chemical derivatization of hydrolysate (amino acids) of the protein; and high-performance liquid chromatography (HPLC) separation, detection, and analysis ofthose derivatized amino acids.The commercially available amino acid analyzers (e.g., Waters Pico-Tag system[Waters Corp., Milford, MA]; GBC AminoMate system [GBC Scientific, Dandenong,Victoria, Australia]) have made amino acid analysis more practical and feasible inroutine protein analysis laboratories. The sensitivity of the analysis has been dramati-cally increased to low picomole levels of proteins, including those low molecularweight (10–20 kDa) ones (low amount of total amino acids analyzed) (1).In this chapter, we describe a 9-fluorenylmethyl oxycarbonyl chloride (FMOC)-based precolumn derivatization amino acid analysis that has been extensively vali-dated (1,2). Although the detailed protocols on the use of the automated GBCAminoMate (GBC Scientific) amino acid analyzer have been described elsewhere (3),here, we emphasize the procedures that are used in a manual operation. Thus, thistechnique can be easily adapted in any laboratory where an HPLC system with a fluo-rescent detector and gradient controller is available.Acid hydrolysis is the first and most important step to release the amino acids fromthe proteins, and it must be carefully controlled in the analysis of mucins. The acidhydrolysis described here recovers 16 amino acids (asparagine and glutamine aredeamidated to their corresponding acids, whereas tryptophan and cysteine are destroyed).During the acid hydrolysis, the carbohydrate side chains on the mucins are degraded.Because of the high carbohydrate content of mucins (up to 90% of the dry weight), thesugars can be caramelized and further charcoaled, and the acid hydrolysis results in ablack residue. This residue appears to precipitate protein/amino acids, interferes with 114 Yan and Packerchromatography, and leads to a significantly lower recovery of all the amino acids,especially serine and threonine. Strong acid, high temperature, and short time acidhydrolysis (12 N HCl at 155°C for 1 h) is generally used in our laboratory for lessglycosylated proteins (1). We have found that weaker acid, lower temperature, andlonger time (ensuring the completion of the hydrolysis) acid hydrolysis (6 N HCl at105°C for 24 h) is a must to obtain reproducible quantitative analysis of mucin gly-coproteins. The hydrolysate of mucin is then derivatized with Fmoc on the α-aminogroups under alkaline conditions. The derivatized amino acids are separated by a C18reversed-phase column and detected by fluorescence. Our routine analyses have shownthat the method described here is reliable and useful for both quantitative andqualitative mucin glycoprotein amino acid analysis.2. Materials2.1. Apparatus2.1.1. Hydrolysis Equipment1. Hydrolysis vessel: the design can be viewed on the World Wide Web at http://www.bio.mq.edu.au/APAF (see Note 1).2. Vacuum pump: Savant Speedvac (Savant Industries, Farmingdale, NY) (or equivalent)with a vacuum gauge.3. Two-way line connection to an argon line and a vacuum pump.4. Autosampler glass vials made from inert Chromacol Gold™ grade glass (Chromacol, cat.no. 02-MTVWG, Herts, UK).2.1.2. HPLC System (seeNote 2)1. LC-pump with ternary gradient controller.2. Fluorescence detector: excitation, λ = 270 nm; detection, λ = 316 nm.3. Sample injector.4. Degasser: alternatively, buffers can be degassed by continuous flow of helium.5. Hypersil C18 reversed-phase column: 150 × 4.6 mm inner diameter 5 µm (Keystone,Bellefonte, PA).6. In-line filter (2 µm) (Upchurch, cat. no. 100-10).2.2. Chemicals2.2.1. Hydrolysis and Derivatization Reagents1. Hydrochloric acid: constant boiling temperature 6 N (Pierce, cat. no. 24309).2. Ultrapure phenol (ICN, cat. no. 800672) kept at 4°C.3. Borate buffer: 250 mM boric acid (analytical reagent grade [AR] grade) in water, adjustedto pH 8.5 with NaOH. Buffer may be kept up to 1 mo at 4°C.4. Fmoc reagent: 4 mg/mL fluorenylmethyl chloroformate (Sigma, cat. no. F0378) in aceto-nitrile (HPLC grade). Reagent may be stored up to 1 wk at 4°C.5. Cleavage reagent: 680 µL of 0.85 M NaOH (AR grade), 150 µL of 0.5 M hydroxylaminehydrochloride (Sigma, cat. no. H2391), and 20 µL of 2-methylthio-ethanol (Sigma, cat.no. M9268). Stock solution can be kept for 1 mo at 4°C. Working cleavage reagent mustbe made freshly prior to each use.6. Quenching reagent: 2 mL of acetic acid (AR grade) and 8 mL of acetonitrile. Amino Acid Analysis of Mucins 1152.2.2. Chromatography Reagents1. Amino acid standards H: protein hydrolysate standard in 0.1 N HCl, containing a solutionof 17 amino acids (Sigma, cat. no. A9781).2. L-hydroxyproline used as internal standard (Sigma, cat. no. H1637).3. Phosphate buffer (2 M): 2 M anhydrous ammonium monohydrogen phosphate (AR grade)solution, adjusted to pH 6.5 with 2 M anhydrous dihydrogen phosphate (AR grade) solu-tion. Buffer may be kept up to 6 mo at 4°C.4. Mobile phase A (30 mM ammonium phosphate [pH 6.5]): Into a 1000 mL volumetricflask, add 15 mL of 2 M phosphate buffer and dilute to volume with mobile phase B.5. Mobile phase B: 15% (v/v) methanol in water.6. Mobile phase C: 90% (v/v) acetonitrile in water.3. Methods3.1. Sample Preparation and Hydrolysis1. Add an aliquot of a solution of a sample of mucin into an autosampler glass vial and dryunder vacuum (see Note 3).2. Place the vials, 400 µL of HCl (6 N), and a crystal of phenol into the bottom of thehydrolysis vessel.3. Assemble the vessel tightly. Connect the vessel to a two-way argon and vacuum line.4. Evacuate the vessel to 3 torr, and flush with argon. Repeat this step twice, and seal thevessel after the third evacuation step (see Note 4).5. Place the vessel in a 105°C oven for 24 h.6. Remove the vessel from the oven and open the vacuum tap immediately within a fumehood (see Note 4).7. Place the vials into a vacuum centrifuge for 10 min to evaporate excess HCl.3.2. Derivatization (see Note 5)1. Dissolve the mucin hydrolysate in 10 µL of 250 mM borate buffer, pH 8.5.2. Add 10 µL of Fmoc reagent, mix, and then wait 1 min.3. Add 10 µL of cleavage reagent, mix, and then wait 4 min.4. Add 10 µL of quenching reagent mix.3.3. Chromatography (see Note 6)1. Set up the fluorescent detector with excitation λ = 270 nm and detection λ = 316 nm.2. The mobile phase is a ternary solution system using the gradient shown in Table 1.3. The stationary phase is a 5 µm Hypersil C18 column that is temperature controlled at38°C. Flow rate is 1.0 mL/min. Allow the column to equilibrate with two gradient runsbefore the sample injection.4. Inject an aliquot (≥5 µL) of each derivatized sample (see Note 6). Run time is 35 min.Allow at least 2 min for pump and column equilibration with the initial composition ofthe mobile phase.5. Figures 1 and 2 show typical chromatograms of the separation of amino acid standardsand hydrolyzed bovine conjunctival mucin (10–20 µg).6. After the run, the peaks can be integrated and the picomoles of each mucin amino acidcalculated by comparison with the peak areas given by the amino acid standard. Since thetotal number of amino acids (or the molecular mass) in a mucin glycoprotein is unknown(or difficult to determine), the actual number of picomoles of mucin protein cannot bedetermined by this analysis. In practice, the amount of mucin can usually be referred to as 116 Yan and PackerTable 1Gradient for the Baseline Separationof 16 Fmoc–Derivatized Amino Acids Within 35 min% Mobile phase A % Mobile phase B % Mobile phase CTime (30 mM ammonium (15% [v/v] (90% [v/v](min) phosphate, pH 6.5) methanol/water) acetonitrile/water)017 68 15117 68 1532 10.8 43.2 4632.05 0 0 10034 0 0 10034.05 17 68 1535 17 68 15Fig. 1. Typical chromatogram of separation of 16 standard Fmoc-amino acids. RFU = rela-tive fluorescence units; Amino acid code: D = aspartic acid, E = glutamic acid, HP = hydroxyproline, S = serine, H = histidine, G = glycine, T = threonine, A = alanine, P = proline,Y=tyrosine, R = arginine, V = valine, M = methionine, I = isoleucine, L = leucine, F =phenylalanine, K = lysine.the amount or ratio of certain amino acids, so a relative comparison in quantitative analy-sis, e.g., carbohydrate analysis, can be done. The amino acid composition of the mucin(%pmol) = pmol of mucin amino acid/total pmol of total amino acids x 100% (see Table 2for an example) (see Note 7). Amino Acid Analysis of Mucins 1174. Notes1. The hydrolysis vessel can be made by any glassblower. It has to withstand high tempera-ture in the presence of strong acid vapor, and hold positive and negative gas pressureduring each hydrolysis run.2. The detection of amino acids can be achieved by any HPLC system. However, if Fmoc isused as the derivatization reagent, a fluorescence detector is essential. The HPLC mustconsist of a ternary gradient controller in order to run the gradient program necessary forthe baseline separation of 16 amino acids. We have investigated different types of C18reversed-phase column and have found that the Hypersil C18 column manufactured byKeystone provides the best separation and resolution. We use the GBC automatedAminoMate HPLC system controlled by GBC WinChrom Windows software that pro-vides an automatic integration of the chromatogram and assignment of peaks (3).3. Sample preparation is an important step in amino acid analysis. Ideally, samples shouldbe dried and free of salts, amines, and detergents. The sample of mucin must be desalted(e.g., by using a size-exclusion desalting column), or the mucin can be dot-spotted ontopolyvinylidene difluoride (PVDF) membrane, or gel separated by electrophoresis andelectroblotted onto PVDF. Note that mucins are often difficult to bind to PVDF. Wilkinset al. (3) have described a detailed protocol for PVDF-bound protein samples.4. During the evacuation and argon flush of the hydrolysis vessel, the bottom of the vesselwill become cold and the acid will boil under vacuum. These are the signs that the vesselis sealed tightly. Following hydrolysis, the vessel should be opened as soon as it isremoved from the oven. Acid condensation inside the vessel is an indication of a com-plete hydrolysis. Handling the vessel requires heat-resistant gloves, and safety gogglesmust be worn at all times.Fig. 2. Typical chromatogram of separation of amino acids from bovine conjunctival mucinhydrolysate. RFU = relative fluorescence units. Amino acid code as defined in Fig. 1. 118 Yan and Packer5. The mucin hydrolysate can be analyzed for its amino acid composition by any otherchemical derivatization procedure and HPLC separation, e.g., phenylisothiocyanatederivatization with ultraviolet detection. If an autosampler is available, the derivatizationprocedure can be automated. Nevertheless, manual derivatization is adequate when thor-ough mixing after the addition of each reagent is practiced.6. The HPLC separation is highly reproducible, requiring no modification of the gradient orconditions from day to day, and only minor maintenance throughout the life of the column,which is usually capable of 800 injections. Wilkins et al. (3) describe detailed trouble-shooting of the chromatography.7. Note that this amino acid analysis does not take into account the amount of cysteine andtryptophan. We are currently developing a method for quantitating cysteine by aminoacid analysis.Table 2Example of Quantitative Calculation of Mucin Amino Acid CompositionBovineconjunctivalStandard mucin %Amino acid (area)a(area) pmolbCompositioncAsx 258,795,760 404,279,488 391 9.6Glx 215,720,688 532,183,264 617 15.1Hydroxy-Pro 249,767,456 243,187,696 243 0.0Ser 209,927,392 426,117,952 507 12.4His 143,969,008 20,205,276 35 0.9Gly 213,371,584 607,125,312 711 17.4Thr 203,866,048 179,105,712 220 5.4Ala 213,553,008 271,715,648 318 7.8Pro 216,618,864 149,426,064 172 4.2Tyr 91,930,856 7,537,291 20 0.5Arg 200,286,320 84,959,272 106 2.6Val 203,002,480 169,595,904 209 5.1Met 144,096,656 29,927,380 52 1.3Ile 202,357,184 132,131,184 163 4.0Leu 201,993,024 270,782,624 335 8.2Phe 206,174,464 104,213,960 126 3.1Lys 292,284,352 109,133,576 93 2.3Total 4077 100aThe on-column amount of each standard amino acid is generally 125 pmol. For example, for a10 µL injection of 50 µM stock solution, on-column pmol of standard amino acid = 50 µM × 10 µL/4,where 4 is the dilution factor in derivatization.bThe pmol of mucin amino acid = peak area of mucin amino acid/peak area of standard aminoacid × 25 pmol × 2, where 2 is the dilution factor from the half of the sample injected.c%pmol = pmol mucin amino acid/total mucin amino acids × 100%, where total mucin amino ac-ids is the sum of mucin amino acids (except cysteine and tryptophan; asparagine and glutamine arerecovered as their acid form: aspartic acid and glutamic acid, respectively). Amino Acid Analysis of Mucins 119AcknowledgmentsThe authors acknowledge support for their research on amino acid analysis throughgrants from the Australia Research Council, Macquarie University Research Grants,National Health and Medical Research Council, and GBC Scientific Equipment(Dandenong, Victoria, Australia). Jun Yan acknowledges financial support through anMUCAB scholarship. The authors also thank Malcom Ball for donating mucin samplesand Dr. Andrew Gooley and Prof. Keith Williams for their support.References1. Yan, J. X., Wilkins, M .R., Ou, K., Gooley, A. A., Williams, K. L., Sanchez, J.-C.,Golaz, O., Pasquali, C., and Hochstrasser, D.F. (1996) Large-scale amino-acid analysisfor proteome studies. J. Chromatogr. 736, 291–302.2. Ou, K., Wilkins, M.R., Yan, J.X., Gooley, A.A., Fung, Y., Sheumack, D., and Wil-liams, K. L. (1996) Improved high-performance liquid chromatography of amino acidsderivatized with 9-fluorenylmethyl chloroformate. J. Chromatogr. 723, 219–225.3. Wilkins, M. R., Yan, J. X., and Gooley, A. A. (1999) 2-DE spots amino acid analysis with9-fluorenylmethyl chloroformate, 2-D Protein Gel Electrophoresis Protocols (Link, A. J.,ed.), Humana, Totowa, NJ, 445–460. . Amino Acid Analysis of Mucins 113113From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield. and Gooley, A. A. (1999) 2-DE spots amino acid analysis with9-fluorenylmethyl chloroformate, 2-D Protein Gel Electrophoresis Protocols (Link, A. J.,ed.),