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Glycoprotein methods protocols - biotechnology
Sequencing Glycopeptides 121121From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ11Identification of Glycosylation Sites in Mucin Peptidesby Edman DegradationNatasha E. Zachara and Andrew A. Gooley1. IntroductionAlthough it is possible to determine and characterize the total carbohydrate profileafter release of the mucin oligosaccharides (usually by β-elimination), it is challeng-ing to assign the sites of glycosylation (macroheterogeneity) and the carbohydrateheterogeneity at a given glycosylation site (microheterogeneity). Typically, the char-acterization of macro- and microheterogeneity has been dependent on the isolation ofsmall peptides with only one glycosylation site. However, this is not possible withhigh molecular weight, heavily glycosylated domains such as those found in mucins.The two methods for determining the sites of glycosylation in proteins are massspectrometry and Edman sequencing. Multiple sites of glycosylation cannot easily bedetected using mass spectrometry; one strategy involves the β-elimination of the car-bohydrate, which results in the conversion of serine to dehydro-alanine and threonineto α-amino butyric acid. These amino acids have unique masses and can be used tomap glycosylation sites (1,2). However, the macro- and microheterogeneity of thecarbohydrates can not be determined unless the peptide has just one glycosylation site.In 1950, Edman sequencing was introduced as a repetitive degradation of proteinswith phenylisothiocyanate (3). In the mid-1960s, the process was automated (4),resulting in a machine where the N-terminal amino acid is derivatized, cleaved, andtransferred to a separate reaction vessel, in which it undergoes a conversion to aphenylthiohydantoin (PTH)-amino acid (Fig. 1). It is the PTH-amino that is separatedby reversed-phase chromatography and detected.Although the modern pulsed liquid sequenator is pmol sensitive, glycosylatedamino acids are only recovered in low yield. Samples are sequenced (Fig. 1) on glass-fiber supports or membranes such as polyvinylidene difluoride (PVDF). Followingcleavage, the released amino acid is transferred from the reaction cartridge to the con-version flask by nonpolar solvents such as ethyl acetate or chlorobutane. The more 122 Zachara and Gooleypolar glycosylated amino acids are not soluble and remain in the support matrix (seeFig. 1, vessel 1) and a blank cycle is observed in the sequencer.An alternative approach is to use solid-phase sequencing to extract glycosylatedamino acids (5). Peptides and proteins are covalently attached to solid supports (6,7),allowing the delivery of polar reagents such as trifluoroacetic acid (TFA) and metha-nol, which facilitates the transfer of glycosylated amino acids. More importantly, ithas been shown that amino acids modified by different carbohydrates have differentretention times on high-performance liquid chromatography (HPLC) (8). Thus, for thefirst time, both macro- and microheterogeneity can be determined for a glycopeptideor glycoprotein in a single experiment (5). This technique has allowed mucin (andmucin-like) glycopeptides and trifluoromethane sulfonic acid (TFMSA)-treated mu-cin tandem repeats to be sequenced through the heavily glycosylated regions and themacroheterogeneity to be assigned and quantitated (9–11).Simple modifications to most modern sequenators facilitate the identification ofglycosylated amino acids (9,11,12). Although it is possible to identify amino acidssubstituted with a monosaccharide using standard programs, the glycosylated aminoacids are not recovered quantitatively. Modifications of the transfer solvents to morepolar reagents, such as TFA or methanol, increase the recovery of glycosylated aminoacids modified by monosaccharides and larger oligosaccharides (degree of polymer-ization [DP]=19). Elsewhere we have published alternative HPLC methods for theseparation of PTH-glycosylated amino acids on a modified Beckman LF3600sequenator (12). These methods were developed for two reasons: (1) to provide a dis-tinct elution window for the PTH-glycosylated amino acids, and (2) to be of lowenough salt concentration for the direct infusion of the PTH-glycosylated amino acidFig. 1. Edman degradation. Schematic version of Edman degradation in the modernsequenator. (PITC, phenylisothiocyanate). Sequencing Glycopeptides 123into an electrospray ionisation mass spectrometer. However, the chromatographydescribed in ref. 12 is not commercially available and must be made in-house. Thefollowing examples from the Hewlett Packard G1000A and the Procise™ from PEBiosystems show HPLC separations (Fig. 2) of PTH-glycosylated amino acidsachieved with commercial kits.2. Materials2.1. Chemicals1. Human Glycophorin A, blood type NN (Sigma, cat. no. G-9266).2. Bovine κ-Casein glycopeptide (Sigma, cat. no. C-2728).3. Sequencing grade TFA.4. HPLC grade solvents, Milli Q water.2.2. Reagents for Adsorption onto Hyperbond1. Hyperbond membranes were from Beckman.2.3. Protein Coupling to Arylamine Membranes (see Note 1)1. Reagents are available in kit form from Beckman and PerSeptive Biosystems (MA; seeNote 2).2. Milli Q water and analytical grade methanol are required for wash steps.2.4. Edman Degradation1. Reagents are obtained in the form of quality-assured sequencing kits, from the manufac-turers of the sequenators.2.5. Apparatus1. Heating blocks at 80°C and 50°C.2. Speed-Vac™ vacuum centrifuge (Savant Instruments, NY)3. Beckman LF3600 protein sequenator, or Hewlett Packard G1000A protein sequencer, orPE Biosystem’s Procise™.3. Methods3.1. Sample PreparationEdman sequencing is a molar-dependent chemistry, and it is necessary to determinehow many moles of glycopeptide are to be sequenced (see Note 3). Samples for Edmandegradation (see Note 4) must be salt free and not contain sialic acid (see Note 5).Samples can be electroblotted (see Note 6) or adsorbed onto Hyperbond or PVDF (seeNote 7), or covalently coupled to an arylamine-derivatized solid support (see Note 8).For qualitative analysis of glycosylation sites, as little as 10 pmol of material isrequired, whereas for quantification of the macro- and microheterogeneity, morematerial is required, depending on the length of the peptide and the variation in thestructures present (see Note 9).3.1.1. Desialylation of Glycopeptides1. Place glycopeptide (5–50 pmol/µL) in salt-free buffer (up to 20% organic modifier [v/v])in a screw-capped polypropylene Eppendorf tube. 124 Zachara and GooleyFig. 2. Glycosylated PTH-amino acid profiles. (A) The glycosylated amino acid window with theelution profiles of PTH-threonine-GalNAc-Gal (black arrowheads) and PTH-threonine-GalNAc (grayarrowheads) from Hewlett Packard G1000A protein sequencer. Standard amino acids are shown forcomparison (threonine, open arrowhead). These amino acids were separated using the manufacturer’ssuggested solvent system and program according to the Program 3.1 chemistry. (B) The glycosylatedamino acid window with the elution profiles of PTH-serine-GalNAc-Gal (black arrowheads) and PTH-serine-GalNAc (gray arrowheads) from Hewlett Packard G1000A protein sequencer. Standard aminoacids are shown for comparison (serine, open arrowhead). These amino acids were separated using themanufacturer’s suggested solvent system and program according to the 3.1 chemistry. (C) Theglycosylated amino acid window with elution profiles of PTH-threonine-GalNAc-Gal (black arrow-heads) and PTH-threonine-GalNAc (gray arrowheads) from the Applied Biosystems Procise. Stan-dard amino acids are shown for comparison (threonine, open arrowhead). These amino acids wereseparated using the manufacturer’s suggested solvent system. Sequencing Glycopeptides 1252. Add an equal volume of 0.2 M TFA.3. Incubate samples at 80°C for 30 min.4. Remove TFA by lyophilization in a vacuum centrifuge (see Note 10).5. Resuspend desialylated glycopeptide in 10–20% (v/v) acetonitrile.3.1.2. ElectroblottingGlycopeptides can be desialylated before or after electrophoresis. Electro-blotted glycopeptides are desialylated as in Subheading 3.1.1. with the follow-ing precautions:1. Prior to addition of TFA, place membranes in the reaction vessel and wet with 10 µL ofmethanol.2. Add enough 0.1 M (not 0.2 M as in Subheading 3.1.1.) TFA to cover the membrane.3. Following incubation at 80°C, remove the membrane from the TFA, wash in Milli Qwater (three times) and dry.3.1.3. Adsorption of Glycopeptides onto Hyperbond or PVDF1. Place a piece of Hyperbond or PVDF (a round membrane is preferable, diameter ~0.5 cm;see Note 11) in a lid of an Eppendorf tube and place on a hot plate at 50°C.2. Wet with 10 µL of methanol.3. When the excess methanol has evaporated, but before the membrane has dried, apply thesample in 10-µL aliquots (see Note 12).4. When all of the sample is applied dry the membrane.3.1.4. Covalent Attachment of GlycopeptidesGlycopeptides are covalently attached to arylamine-derivatized membranes via theactivation of peptide carboxyl groups using water-soluble EDC (see Note 13). Cova-lent coupling of the peptide to arylamine-derivatized membrane is as described in thekit user’s guide.1. Place an arylamine-derivatized membrane in an Eppendorf tube lid at 50°C.2. Apply samples in 10-µL aliquots, allowing the membrane to come to near dryness betweeneach aliquot (see Note 14).3. Dry the membrane after all of the sample has been applied.4. Mix approx 1 mg of EDC in 50 µL of reagent attachment buffer (see Note 15) and care-fully pipet 10–50 µL onto the arylamine-derivatized membrane.5. Incubate the sample at 4°C for 30 min (see Note 16).6. Wash the membrane alternately in 1 mL of methanol and 1 mL of Milli Q water threetimes and dry.3.2. Edman Sequencing3.2.1. Glycopeptides Electroblotted or Adsorbed onto Hyperbond or PVDFGlycopeptides adsorbed onto PVDF or Hyperbond can be sequenced with conven-tional Edman degradation (Program 3.1 PVDF on the Hewlett Packard G1000A or thePVDF routine for the blot cartridge on the Procise, or a modified program 40 recom-mended by Beckman for PVDF on the Beckman LF series). While amino acids modi-fied by monosaccharides will be identified, their recovery (as well as that of the largeroligosaccharides) is not quantitative. 126 Zachara and Gooley3.2.2. Glycopeptides Covalently Attached to MembranesCovalently attached glycopeptides can be sequenced using a modified HewlettPackard G1000A routine 3.1 PVDF that includes methanol rather than ethyl acetatefor the extraction of the cleaved amino acid from the cartridge (Fig. 1, vessel 1; seeNote 17) to the converter (Fig. 1, vessel 2; see Note 18).4. Notes1. The arylamine-coated membranes are stable at –20°C; the N-ethyl-N'-dimethylamino-propylcarbodiimide (EDC reagent is stable at 4°C in a dry environment, and the buffer isstable at 4°C.2. Arylamine-derivatized membranes, Sequelon AA™, were originally produced byMilligen Biosearch, a division of Millipore. Although Millipore no longer manufacturesthese membranes, both PerSeptive Biosystems and Beckman supply this product.3. Quantitation of glycopeptides is quite difficult, and we recommend that 10% of the samplebe first sequenced using the standard program prior to glycosylation site mapping.4. Mucins must be digested extensively by proteases and the glycopeptide purified,desialylated (see Note 5), and desalted before sequencing. For heavily glycosylated pep-tides substituted with large oligosaccharides it may be necessary to treat with TFMSA(13) or glycosidases before sequencing.5. There are two principle reasons for desialylating glycopeptides:a. If the peptide is to be covalently bound to an arylamine-derivatized support, the sialicacid must be removed to prevent the formation of an amide bond between the car-boxyl group of the sialic acid and the amine of the support. Although the alternativeof using diisothiocyanate-derivatized membranes is possible, we have found them oflittle practical use, since the peptides must contain lysine, preferably at the C-termi-nus, and this residue is uncommon in mucins.b. For glycopeptides adsorbed onto Hyperbond or PVDF supports, we have observed poorrecovery of sialylated glycosylated amino acids. They elute as a series of multiple peaksand, in many cases, coelute with standard amino acids (unpublished results).6. Electroblotting is not a usual method for the preparation of mucin glycopeptides sincethey are large in apparent molecular weight and are rarely separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, if glycopeptides canbe separated by SDS-PAGE, the glycopeptides can be electroblotted onto hydrophobicmembranes such as Hyperbond or PVDF and stained with amido black.7. Hyperbond performs better in this application than PVDF.8. Covalent coupling gives a quantitative recovery of amino acids modified by more thanone carbohydrate residue. Other methods only identify an amino acid modified by onecarbohydrate residue.9. Because mucin glycopeptides are usually of very high molecular weight, an initial yieldof 10 pmol in the sequenator represents approx 100 µg of a 200-kDa glycopeptide. Quan-titating the carbohydrate component (see Notes 3 and 8) and the length of sequenceobtained is dependent on the number and distribution of proline residues. Proline cleaveswith reduced efficiency in Edman sequencing (Fig. 1, vessel 1), and a significant lag isintroduced when several prolines occur in a cluster and more than one signal is detectedin each cycle.10. Freeze-drying mucins is not recommended because the solid is often poorly soluble. Thesample should not be taken to complete dryness. Sequencing Glycopeptides 12711. Round pieces of PVDF or Hyperbond have better surface tension and allow larger aliquotsto be loaded. Alternatively, a strip of PVDF or Hyperbond can be used, approx 2 × 10 mm.Care must be taken when loading to maintain surface tension on the strip.12. Membranes should not be allowed to dry between application of sample aliquots.13. Although small peptides couple well, the high molecular weight of mucin glycopeptidesreduces the efficiency of coupling to ~5%.14. Amine buffers such as Tris interfere with coupling to arylamine and should be avoided.15. The EDC reagent should be mixed with the reaction buffer just prior to coupling.16. Incubation at 4°C increases the initial yield (7).17. To incorporate a methanol extraction into the routine, methanol is placed in an unusedbottle position, and solvent delivery from this bottle is substituted for ethyl acetate duringthe extraction procedure.18. Poor yields of aspartic and glutamic acid will be observed with arylamine-coupled peptidesor proteins because of the coupling of the χ- and δ-carboxyl groups to the membrane.AcknowledgmentsNEZ was supported by an Australian Postgraduate Award. AAG acknowledges thesupport of the National Health and Medical Research Council. The authors would liketo acknowledge the support and useful comments of Dr. Nicolle Packer and Prof. KeithWilliams (Macquarie University).References1. Rademaker, G. J., Haverkamp, J., and Thomas-Oates, J. (1993) Determination of glyco-sylation sites in O-linked glycopeptides: a sensitive mass spectrometric protocol. Organ.Mass Spectrom. 28, 1536–1541.2. Gries, K. D., Hayes, B. K., Comer, F. I., Kirk, M., Barnes, S., Lowary, T. L., and Hart, G.W. (1996) Selective detection and site-analysis of O-GlcNAc-modified glycopeptides byβ-elimination and tandem electrospray mass spectrometry. Anal. Biochem. 234, 38–49.3. Edman P. (1950) Method for the determination of the amino acid sequence in peptides.Acta. Chem. Scand. 4, 283–293.4. Edman, P and Begg, G., (1967) A protein sequencer. Eur. J. Biochem. 1, 80–91.5. Gooley, A. A., Classon, B. J., Marschalek, R., and Williams, K. L. (1991) Glycosylationsites identified by detection of glycosylated amino acids released from Edman degrada-tion: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation. Biochem.Biophys. Res. Commun. 178, 1194–1201.6. Laursen, R. A. (1971) Solid-phase Edman degradation: an automated peptide sequencer.Eur. J. Biochem. 20, 89–102.7. Laursen, R. A., Lee, T. T., Dixon, J. D., and Liang S.-P. (1991) Extending the perfor-mance of the solid-phase sequenator, in Methods in Protein Sequence Analysis (Jornvall,H., Hoog, J.-O., and Gustavsson, A.-M., eds.), Birkhauser Verlag, Switzerland, pp. 47–54.8. Gooley, A. A. and Williams, K. L. (1997) How to find, identify and quantitate the sugarson proteins. Nature 358, 557–559.9. Muller, S., Goletz, S., Packer, N., Gooley, A., Lawson, A. M., and Hanisch, F. G. (1997)Localisation of O-glycosylation sites on glycopeptide fragments from lactation-associatedMUC1: all putative sites within the tandem repeat are glycosylation targets in vivo. 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(1992) A novel approach for chemicallydeglycosylating O-linked glycoproteins: the deglycosylation of submaxillary and respira-tory mucins. Biochemistry 31, 639–648. . profiles of PTH-threonine-GalNAc-Gal (black arrow-heads) and PTH-threonine-GalNAc (gray arrowheads) from the Applied Biosystems Procise. Stan-dard amino acids. glycosylated amino acids released from Edman degrada-tion: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation. Biochem.Biophys. Res. Commun.