Mass Spectrometric Determination of O-Glycosylation Sites Using β

Feb 15, 2001 - methylamine: Δ indicates loss of acetyl group (r42 Da) from the. GalNAc residue; asterisk (*) indicates a mass increment of +13 Da on ...
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Anal. Chem. 2001, 73, 1263-1269

Mass Spectrometric Determination of O-Glycosylation Sites Using β-Elimination and Partial Acid Hydrolysis Ekaterina Mirgorodskaya,†,§ Helle Hassan,‡ Henrik Clausen,‡ and Peter Roepstorff*,†

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark, and Faculty of Health Science, School of Dentistry, University of Copenhagen, DK-2200, Copenhagen N, Denmark

The present study demonstrates that treating O-glycosylated peptides with methylamine vapor followed by partial acid hydrolysis is an effective means for locating Oglycosylation site(s). The reaction with methylamine transforms the glycosylated Ser and Thr residues into stable methylamine derivatives with a mass increment of +13 Da relative to nonglycosylated Ser and Thr residues. Peptide sequencing based on partial acid hydrolysis followed by mass spectrometric analysis or in favorable cases by CID-MS/MS enables the determination of the formerly O-glycosylated sites. Several different monosaccharides can form O-glycosidic linkage to the polypeptide chain. Addition of N-acetylgalactosamine (GalNAc) to Ser and Thr is one of the most common types of mammalian O-glycosylation. This glycosidic linkage is characteristic of ephithelian mucines and is, therefore, often referred to as mucin-type glycosylation. Mass spectrometric analysis is a commonly used technique for characterization of protein posttranslational modifications. However, due to structural properties of mucin-like glycoproteins, determination of the glycosylated sites often is a challenging task. Mucin-type glycosylation frequently occurs in regions with high densities of Ser, Thr, Gly, and Pro residues. Because none of these residues represents cleavage sites for commercially available proteases, determination of the utilized sites based on mass spectrometric peptide mapping is often not possible. A number of strategies based on tandem MS for the localization of the glycosylated residues has been reported,1-8 * Corresponding author: Phone: +45 65 50 24 04. Fax: +45 65 93 26 61. E-mail: [email protected]. † University of Southern Denmark. ‡ University of Copenhagen. § Present address: Mass Spectrometry Resource, Boston University, 715 Albany St. R-806, Boston, MA 02118. (1) Goletz, S.; Leuck, M.; Franke, P.; Karsten, U. Rapid Commun. Mass Spectrom. 1997, 11, 1387-1398. (2) Goletz, S.; Thiede, B.; Hanisch, F. G.; Schultz, M.; Peterkatalinic, J.; Muller, S.; Seitz, O.; Karsten, U. Glycobiology 1997, 7, 881-896. (3) Mu ¨ ller, S.; Goletz, S.; Packer, N.; Gooley, A.; Lawson, M.; Hanisch, F.-G. J. Biol. Chem. 1997, 272, 24780-24793. (4) Settineri, C. A.; Medzihradszky, K. F.; Masiarz, F. R.; Burlingame, A. L.; Chu, C.; George-Nascimento, C. Biomed. Environ. Mass Spectrom. 1990, 19, 665-676. (5) Medzihradszky, K. F.; Gillece-Castro, B. L.; Settineri, C. A.; Townsend, R. R.; Masiarz, F. R.; Burlingame, A. L. Biomed. Environ. Mass Spectrom. 1990, 19, 777-781. 10.1021/ac001288d CCC: $20.00 Published on Web 02/15/2001

© 2001 American Chemical Society

including both PSD and CID analysis. The major limitation of PSD and CID for the determination of the utilized glycosylation sites on glycopeptides is the glycosidic bond lability. Furthermore, PSD and CID produce relatively selective polypeptide backbone cleavages, depending on the nature of the neighboring amino acids; therefore the success of MS/MS analysis can be limited by the nature of the mucin-type peptides containing clusters of Ser and Thr residues and, in addition, a high density of Gly residues. The recently reported new MS/MS method of electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT-MS)9-11 is an excellent technique for site-specific determination of O-glycosylation. It produces abundant polypeptide fragments, the sole exception being X-P (X being any amino acid), without loss of labile post-translational modifications, such as O-linked glycans12 and the carboxyl group in γ-carboxyglutamic acid.13 However, at the present moment, FT-MS is not a commonly available in biological laboratories and, furthermore, ECD is not available on all existing FT-MS instruments. Therefore, it is still of interest to develop alternative methods based on readily available instrumentation. Recently, as part of an ongoing study of the acceptor-site specificity of the GalNAc-transferases,14 we explored the potential of acid hydrolysis for determination of mucin-type glycosylation sites in glycopeptides.15 Due to the high density of the residues that provide efficient acid cleavage sites (Ser, Thr, and Gly) in the mucin-type peptides, the hydrolysis resulted in extensive polypeptide bond cleavages, allowing differentiation among po(6) Kieliszewski, M. J.; Neill, M. O.; Leykam, J.; Orlando, R. J. Biol. Chem. 1995, 270, 2541-2549. (7) Juge, N.; Andersen, J. S.; Tull, D.; Roepstorff, P.; Svensson, B. Protein Expression Purific. 1996, 8, 204-214. (8) Hanisch, F.-G.; Green, B. N.; Bateman, R.; Peter-Katalinic, J. J. Mass Spectrom. 1998, 33, 358-362. (9) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (10) Kruger, N. A.; Zubarev, R. A.; Carpenter, B. K.; Kelleher, N. L.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 183, 1-5. (11) Kruger, N. A.; Zubarev, R. A.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 187, 787-793. (12) Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Anal. Chem. 1999, 71, 4431-4436. (13) Kelleher, R. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Anal. Chem. 1999, 71, 4250-4253. (14) Clausen, H.; Bennett, E. P. Glycobiology 1996, 6, 635-646. (15) Mirgorodskaya, E.; Hassan, H.; Wandall, H. H.; Clausen, H.; Roepstorff, P. Anal. Biochem. 1999, 269, 54-65.

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tential glycosylation sites. However, due to the partial cleavage of the glycosidic bond, it was observed that as the number of utilized glycosylation sites increased, so did the complexity of the hydrolysates, which consequently complicated the data interpretation for multiple glycosylated peptides. Therefore, further refinement of the described procedure was necessary. One way is to replace the glycans with a group that will be completely stable under acid hydrolysis conditions. Having a distinct mass, a derivative of formerly glycosylated residues facilitates the identification of the glycosylated sites. Alkaline β-elimination with NaOH is a commonly used chemical method for releasing O-linked oligosaccharides from glycopeptides. Unique amino acid residues formed from the formerly O-glycosylated Ser and Thr residues can be used for determination of glycosylation sites.16,17 Commonly used procedures, however, require long reaction times to obtain sufficient cleavage of the O-glycosidic bond and often require additional purification procedures. Sample contamination can be avoided by the use of volatile reagents that can easily be removed from the samples by evaporation. The application of ammonia for the β-elimination reaction has been reported to result in complete deglycosylation after incubation for 4 h at 37 °C.18 β-Elimination of the O-glycosidic bond using NH3 followed by tandem mass spectrometric analysis for identification of modified Ser and Thr residues has been recently reported;19 however, in our hands, these procedures did not result in quantitative release of the glycans, and several different products were obtained as result of the reactions. This led us to investigate reagents other than NaOH or NH3. We here present a method for the assignment of O-glycosylated sites in glycopeptides after quantitative β-elimination of O-glycosidic bonds in a vapor of methylamine. Complete deglycosylation is achieved in 2-4 h, during which time O-glycosylated Ser and Thr residues are converted into stable methylamine derivatives. These derivatives are stable under CID conditions as well as during partial acid hydrolysis, and interpretation of the resulting spectra is considerably simplified. MATERIALS AND METHODS In Vitro Glycosylation of Peptides. Synthetic mucin-type GalNAc-glycosylated peptides were glycosylated in vitro using recombinant polypeptide GalNAc transferases.20,21 Deglycosylation. Glycosylated peptides (20-100 pmol) were lyophilized in 650-µL PCR tubes (BioScience, Inc.). The tubes were placed in a reaction chamber (22 mL glass vial with a mininert valve) containing 150 µL of 40% NH2CH3 (aq) or 25% NH3 (aq). The reaction vial was flushed with argon and incubated for 1-4 h at 70 °C. Traces of solvent were removed in a vacuum centrifuge. (16) Rademaker, G. J.; Haverkamp, J.; Thomas-Oates, J. E. Org. Mass Spectrom. 1993, 28, 1536-1541. (17) Greis, K. D.; Hayes, B. K.; Comer, F. I.; Kirk, M.; Barnes, S.; Lowary, T. L.; Hart, G. W. Anal. Biochem. 1996, 234, 38-49. (18) Pittenauer, E.; Schmid, E. R.; Allmaier, G.; Pfanzagl, B.; Loffelhardt, W.; Fernandez, C. Q.; Depedro, M. A.; Stanek, W. Biol. Mass Spectrom. 1993, 22, 524-536. (19) Rademaker, G. J.; Pergantis, S. A.; Bloktip, L.; Langridge, J. I.; Kleen, A.; Thomas-Oates, J. E. Anal. Biochem. 1998, 257, 149-160. (20) Sørensen, T.; White, T.; Wandall, H. H.; Kristensen, A. K.; Roepstorf, P.; Clausen, H. J. Biol. Chem. 1995, 270, 24166-24173. (21) Wandall, H. H.; Hassan, H.; Mirgorodskaya, E.; Kristensen, A. K.; Roepstorff, P.; Bennett, E. P.; Nielsen, P. A.; Hollingsworth, M. A.; Burchell, J.; TaylorPapadimitriou, J.; Clausen, H. J. Biol. Chem. 1997, 272, 23503-23514.

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Acid Hydrolysis. Glycosylated and deglycosylated peptides (20-50 pmol) were lyophilized in Eppendorf vials. The tubes were placed in a reaction vial containing 100 µL of 20% pentafluoropropionic acid (PFPA). The vial was flushed with argon and subsequently evacuated to 1 mbar. Samples were hydrolyzed for 60-90 min at 90 °C. Traces of solvent were removed in a speedvac. Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Mass Spectrometry (MS). MALDI mass spectra were acquired in linear and reflector mode on a VoyagerElite Biospectrometry Workstation (PerSeptive Biosystems Inc., Framingham, MA) equipped with delayed extraction. Mass spectra were, unless otherwise stated, externally calibrated. Glycosylated peptides prior to and after β-elimination were dissolved in 0.1% trifluoroacetic acid (TFA) to a concentration of 500 fmol/µL, and prepared for MALDI-MS analysis by mixing 0.5 µL of sample solution with 0.5 µL of matrix solution [2,5-dihydroxybenzoic acid (DHB), 10 mg/mL; acetonitrile/0.1% TFA, 1/2 (v/v)] directly on the target. The hydrolyzed samples were dissolved in 0.1% TFA to a concentration of 1-4 pmol/µL, based on the initial amount of peptide subjected to hydrolysis. Samples were prepared for MALDI-MS analysis using nanoscale reversed-phase columns and DHB as a matrix according to a previously described procedure.22 The protein analysis software GPMAW (GPMAW (htpp:// www.welcome.to/gpmaw; Lighthouse data, Odense, Denmark) was used for data interpretation. The observed peptide masses were searched against the theoretically calculated masses for all possible hydrolytic peptide fragments without, as well as with, mass increments corresponding to either attached monosacharide residue(s) or addition of methylamine group(s) at the glycosydic bond(s). Electrospray (ESI) Tandem Mass Spectrometry (MS/MS). ESI mass spectra were acquired on an Esquire ion trap (BrukerFranzen Analytik GmbH) equipped with a nanoelectrospray source. MS/MS spectra were acquired using He as the collision gas. Deglycosylated peptides were dissolved in water/methanol, 1/1 (v/v), to a final concentration of approximately 10 pmol/µL, and 2µL aliquots were used for ESI-MS analysis. RESULTS β-Elimination Using Ammonia and Methylamine. A number of in vitro glycosylated mucin-derived peptides were used for the comparison of β-elimination using ammonia and methylamine. During the β-elimination-addition reaction, O-glycosylated Ser and Thr residues are converted into their stable derivatives (Figure 1), which facilitates their localization in the peptide sequence. Because methylamine is a stronger base than ammonia, the efficiency of β-elimination in a vapor of methylamine was expected to be higher. This was confirmed by comparison of β-elimination using ammonia and methylamine under the same reaction conditions. In vitro glycosylated mucin-derived peptide Muc 1a′ (AHGVTSAPDTR) has three potential glycosylation sites underlined in the sequence and one N-acetylgalactosamine residue (GalNAc) attached to the polypeptide chain. The glycopeptide was incubated with 25% ammonia and 25% methylamine at 70 °C for 2 h. The reactions were monitored using MALDI-TOFMS (Figure (22) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105-116.

Figure 1. β-Elimination of glycans from glycosylated Ser and Thr using a vapor of ammonia/methylamine results in the addition of ammonia/methylamine to the intermediates dehydroalanine (Dha) and dehydrobutyric acid (Dhb), respectively. The resulting stable amino acid derivatives have a mass difference of -1 Da (addition of ammonia) and +13 Da (addition of methylamine) as compared to unmodified Ser/Thr, which facilitates their identification in the sequence. M is the molecular weight of the nonglycosylated peptide.

2). The results indicated that the rate of β-elimination is considerably higher when using methylamine, as compared to ammonia. Incubation of Muc1a′ peptide in a vapor of methylamine at 70 °C for 2 h resulted in the complete deglycosylation of the peptide and a concomitant transformation of the formerly O-glycosylated residue into its stable methylamine derivative (M + 13, where M is the molecular weight of the nonglycosylated peptide). Incubation of the glycosylated peptide in a vapor of ammonia under the same conditions resulted in just partial deglycosylation of the peptide. Three products corresponding to intact glycopeptide (M + GalNAc), the elimination reaction product (M - 18), and an addition reaction product (M - 1) were detected by MALDITOFMS. This indicates that both elimination and addition reactions are less efficient when ammonia is used to release the O-linked glycan. The efficiency of the elimination-addition reaction was found to be different for glycosylated Ser and Thr residues. The reaction was significantly faster for glycosylated Ser than for glycosylated Thr, as demonstrated with peptides PPDAATAAPLR and PPDAASAAPLR in Figure 3. Incubation of the peptides in a vapor of 40% methylamine at 70 °C for 1 h resulted in quantitative deglycosylation of the Ser-containing peptide and only partial deglycosylation of the Thr-containing peptide. Quantitative release of GalNAc from the Thr-containing peptide required prolongation of the reaction time up to 2 h. The efficiency of the elimination-addition reaction strongly depends on the reaction temperature. It was found that the incubation of the mucin-type glycosylated peptides with a vapor

Figure 2. MALDI mass spectra of monoglycosylated peptide Muc1a′ (a) prior to and after β-elimination in vapor of (b) ammonia and (c) methylamine for 2 h at 70 °C. The observed reaction products are labeled according to Figure 1.

Figure 3. MALDI-TOF mass spectra of glycosylated (a) PPDAATAAPLR and (b) PPDAASAAPLR peptides, after incubation with 40% methylamine, at 70 °C for 1 h. The observed reaction products are labeled according to Figure 1.

of 40% methylamine at 70 °C resulted in quantitative deglycosylation in 1-3 h for all peptides included in the study. Further increase of the reaction temperature resulted in undesirable side reactions, such as polypeptide chain cleavages and methylamidation of the C-termini. It was, as well, observed that long reaction times (>3 h) in some cases resulted in polypeptide backbone cleavage of peptides, with concomitant transmethylamidation of the newly formed C-termini. A strong tendency for polypeptide bond cleavage was observed for the Gly-Ser bond when Ser was a nonglycosylated residue. In addition, polypeptide bond cleavages Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

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Figure 4. ESI ion-trap MS/MS spectrum of the methylamine derivative of the formerly monoglycosylated peptide Muc1a′. All fragment ions containing Thr5 appear in the spectrum as M + 13 indicated by an asterisk (*), identifying Thr5 as the glycosylated residue.

were observed when clusters of nonglycosylated Ser residues were present in the sequence. Therefore, it was concluded that quantitative removal of the glycans, without backbone cleavages, requires optimization of the reaction time for each individual sample. Long incubation times should be avoided when the abovementioned labile bonds are present in the sequence. Incubation of mucin-type glycopeptides in a vapor of 40% methylamine (aq) at 70 °C for 2 h was found to be a good starting point for optimization when a quantitative release of glycan is desired. Site Specific Identification of the Formerly Glycosylated Residues in Peptide Sequence. CID-MS/MS of the Methylamine Derivatives. Optimization for quantitative release of the glycans prior to location of the formerly glycosylated residues can be avoided if the ion representing the completely deglycosylated peptide is isolated and subjected to CID. To evaluate feasibility of CID experiments for identification of modified residues, deglycosylated peptides after β-elimination with methylamine were analyzed by CID-MS/MS on the ion trap mass spectrometer equipped with a nano-ESI source. The ESI spectrum of monoglycosylated peptide Muc1a′ after β-elimination showed a mass increment of +13 as compared to the mass of the nonglycosylated peptide, which confirmed the presence of a single glycosylated residue on the polypeptide chain. The doubly charged ion of the deglycosylated peptide Muc1a′ was isolated for CID experiment. The resulting CID spectrum is shown in Figure 4. Among the identified CID fragments, all fragment ions containing Thr5 appeared in the spectrum as M + 13, identifying Thr5 as the glycosylated residue. This indicated that the generated methylamine derivative of Thr remained stable under CID conditions. However, CID experiments using the ion trap for larger peptides did not always allow unambiguous identification of all modified residues. This is mainly due to a preferential fragmentation at X-P and D-X bonds (X being any amino acid residue) upon CID, which results in a low abundance or absence of fragment ions corresponding to cleavages of peptide bonds within Ser and Thr clusters. Furthermore, we were not able to assign all of the observed fragment ions by reported peptide fragmentation mechanisms, so that further optimization will be needed to identify the cleavage site. Partial Acid Hydrolysis of Methylamine Derivatives. We have previously demonstrated that acid hydrolysis can be optimized to generate extensive polypeptide bond cleavages with only partial carbohydrate loss; however, the partial loss of glycans 1266 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

Figure 5. MALDI mass spectra of the hydrolysate of glycosylated peptide Muc1a′ (a) prior to and (b) after β-elimination in a vapor of methylamine: ∆ indicates loss of acetyl group (-42 Da) from the GalNAc residue; asterisk (*) indicates a mass increment of +13 Da on the given hydrolytic fragment.

during hydrolysis increases the heterogeneity of the hydrolysates and renders spectra interpretation for the hydrolysates of glycopeptides with multiple sites difficult. Substitution of the glycan by a group, stable under the hydrolysis conditions, considerably reduces the heterogeneity of the resulting hydrolysates and simplifies their interpretation. Mass spectra of hydolysates of glycosylated and deglycosylated peptide Muc1a′ are shown in Figure 5. Identification of the glycosylated site as Thr5 was possible on the basis of both samples, but the reduced sample heterogeneity simplified the interpretation of the spectrum for the sample that was obtained by acid hydrolysis of the deglycosylated peptide. The mass increment of +13 Da on the given hydrolytic fragment, as compared to the calculated mass based on the sequence of the nonglycosylated peptide, unambiguously identified the presence of the glycosylated site. In the case of acid hydrolysis applied directly to the glycosylated peptide, each glycosylated hydrolytic fragment was identified by the presence of peak pairs in the spectrum corresponding to glycosylated and nonglycosylated hydrolytic fragments resulting from the partial cleavage of the glycosidic bond during hydrolysis. The assignment of the abovementioned pairs was confirmed by a peak 42 Da below those for the glycosylated hydrolytic fragments caused by the loss of the acetyl group from the GalNAc residue.15 Mucin-derived peptide PARVVTSAPDTSAAPG, Mr 1495.8 Da, has four potential glycosylation sites underlined in the sequence. MALDI-MS analysis of the peptides after in vitro glycosylation with GalNAc-T1, -T2, and -T4 transferases showed a mass increment of 406 Da for each of the glycosylated peptides, as compared to the calculated mass based on the sequence of the nonglycosylated peptide corresponding to the presence of two

Figure 6. (a) MALDI mass spectrum of a glycosylated peptide, PARVVTSAPDTSAAPG, after hydrolysis with 20% PFPA at 90 °C for 1 h: ∆, loss of acetyl group (-42 Da) from the GalNAc residue. (b) The observed N-terminal sequence ladder for the glycosylated PARVVTSAPDTSAAPG peptide. The peptide has four potential glycosylation sites underlined in the sequence and two GalNAc groups attached to the polypeptide chain. The glycosylated hydrolytic fragments were observed as fully glycosylated (+2 GalNAc), partially deglycosylated (+1 GalNAc), and nonglycosylated (NG) products due to partial cleavage of the glycosidic bond. The observed hydrolytic fragments gave unambiguous identification of Thr6 as the glycosylated site and an indication of Ser12 as the second glycosylated site. Single dagger indicates calculated monoisotopic masses based on the amino acid sequence; two daggers, measured monoisotopic masses after subtraction of the ionizing proton.

GalNAc groups attached to the polypeptide chain (not shown). Acid hydrolysis of the peptide in vitro glycosylated with GalNAcT2 followed by MALDI-TOF analysis allowed unambiguous identification of Thr6 as glycosylated and Ser7 as nonglycosylated residues (Figure 6). The presence of hydrolytic fragments1-11 carrying only one GalNAc group1-12 and carrying two GalNAc groups indicated that Ser12 is a likely candidate for the second glycosylated site, but due to low abundancies of these hydrolytic fragments, it was difficult to make an unambiguous identification of Ser12 as the second utilized site. β-Elimination using 40% methylamine at 70 °C for 2 h resulted in complete deglycosylation of the peptide glycosylated with GalNAc-T2 transferase. In agreement with the presence of two glycosylated sites, the observed mass after β-elimination showed a mass increment of +26 Da (1521.97 Da), as compared to the one based on the sequence of the nonglycosylated peptide. Acid hydrolysis applied to the deglycosylated peptide generated a N-terminal sequence ladder that was the same as in the case of the glycosylated peptide (Figure 7). The presence or absence of modification on a given Ser/Thr residue unambiguously identified Thr6 and Ser12 as the glycosylated residues. The interpretation of the obtained spectrum was considerably simplified due to reduced sample heterogeneity.

Figure 7. (a) MALDI mass spectrum of the PFPA hydrolysate of a deglycosylated peptide PARVVTSAPDTSAAPG: asterisk (*) indicates a mass increment of +13 Da; two asterisks (**), +26 Da, on the given hydrolytic fragment, as compared to the mass of nonmodified peptide. (b) The observed N-terminal sequence ladder for the methylamine derivative of the peptide PARVVTSAPDTSAAPG. Potential glycosylation sites are underlined in the sequence. The observed hydrolytic fragments allowed unambiguous identification of Thr6 and Ser12 as the glycosylated sites. Single dagger indicates calculated monoisotopic masses based on the amino acid sequence; two daggers, measured monoisotopic masses after subtraction of the ionizing proton.

The same glycosylated sites were identified for GalNAc-T1 and -T4 transferases. The synthetic mucin-derived peptide AAPGSTGPPARVVTSAPD, Mr 1649.8 Da, was in vitro glycosylated with GalNAc-T2 transferase. The peptide has four potential glycosylation sites underlined in the sequence. MALDI-MS analysis of the in vitro glycosylated peptide indicated incorporation of three GalNAc residues by GalNAc-T2 transferase. Complete deglycosylation of the peptide was achieved after its incubation in vapor of 40% methylamine at 70 °C for 2.5 h. In agreement with the presence of three glycosylated sites, the observed mass after β-elimination showed a mass increment of +39 Da (1688.89 Da), as compared to the one based on the sequence of the nonglycosylated peptide. Acid hydrolysis of the deglycosylated peptide generated a short N-terminal sequence ladder (four amino acid residues) and two short partially overlapping C-terminal sequence ladders (Figure 8). The N-terminal sequence ladder showed Ser15 to be nonmodified residue and, therefore, was sufficient to identify Ser5, Thr6, and Thr14 as the glycosylation sites. The presence of modified residues in position Ser5 and Thr6 was further confirmed by observed C-terminal sequence ladders initialized by favorable acid hydrolysis cleavages at Gly and Asp residues. Analytical Chemistry, Vol. 73, No. 6, March 15, 2001

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Figure 8. (a) MALDI mass spectrum of the PFPA hydrolysate of a deglycosylated peptide AAPGSTGPPARVVTSAPD. Single, double, and triple asterisks (*, **, and ***) indicate a mass increment of +13, +26, and +39 Da, respectively, on the given hydrolytic fragment, As compared to the mass of nonmodified peptide. (b) The observed degradation products of the methylamine derivative of the peptide AAPGSTGPPARVVTSAPD. The peptide has four potential glycosylation sites underlined in the sequence. The observed short N-terminal sequence ladder allowed unambiguous identification of Ser5, Thr6, and Thr14 as the glycosylated sites. Single dagger indicates calculated monoisotopic masses based on the amino acid sequence; two daggers, measured monoisotopic masses after subtraction of the ionizing proton.

DISCUSSION The present study demonstrates that the combination of β-elimination in vapor of methylamine with partial acid hydrolysis is an effective means for the determination of O-glycosylated site(s) in glycopeptides. The described results are part of an ongoing study of the acceptor-site specificity of the GalNAc-transferases, a family of enzymes catalyzing the addition of the first Nacetylgalactosamine residue to the polypeptide chain.14 Characterization of GalNAc-transferase specificities requires determination of the utilized sites after in vitro glycosylation of acceptor peptides. Previously, we demonstrated that partial acid hydrolysis in combination with MALDI-TOFMS analysis enables localization of the glycosylated sites.15 During this study, it was observed that partial loss of glycan during hydrolysis increased the complexity of the mass spectra and, consequently, decreased the detection sensitivity due to multiple peaks for each hydrolytic fragment. The latter can be crucial for the unambiguous assignment of glycosylation on low abundance hydrolytic fragments. To overcome this problem, we have attempted to substitute the GalNAc residue with another group that is stable under the acid hydrolysis conditions. This group should result in a distinct mass increment to enable localization of the formerly glycosylated sites. Furthermore, to avoid a separation step prior to acid hydrolysis, the reaction using 1268

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volatile reagents and allowing quantitative release of glycans would be advantageous. Both methylamine and ammonia are volatile reagents that allow the release of O-glycans by β-elimination. Methylamine was found to be superior to amonnia, because the latter required considerably longer reaction times and did not always allow quantitative release of O-glycans. We have assumed that the reaction mechanisms are the same for both ammonia and methylamine and that the improved elimination and addition reactions obtained with methylamine are due to its stronger basicity. However, a different reaction mechanism by going from ammonia to methylamine cannot be excluded. Another advantage of using methylamine is that Ser and Thr residues are converted into methylamine derivatives, resulting in a readily detectable mass increment of +13Da, as compared to mass a decrement of only 1Da when using ammonia. For β-elimination with both reagents, it was observed that long reaction times might result in polypeptide backbone cleavage, with concomitant modification of the newly formed C-termini (transamidation in the case of ammonia and transmethylamidation in the case of methylamine). The observed polypeptide backbone cleavages took place preferentially at the nonglycosylated Ser residues. Therefore, long incubation times should be avoided when clusters of Ser residues are present in the sequence to avoid the polypeptide bone cleavages. Although quantitative removal of the glycans without polypeptide backbone cleavage required optimization of the reaction time in each individual case, predictions of the optimal reaction time can be made based on the primary sequence of the peptide and the number of glycosylated residues. In general, for mucin-type glycopeptides, a good starting point is incubation in a vapor of 40% methylamine (aq) at 70 °C for 2 h. To identify the position of the modified residues in the peptide sequence, deglycosylated peptides were further analyzed using a CID-based MS/MS technique as well as partial acid hydrolysis followed by MALDI-TOF MS analysis of the resulting mixtures. Direct MS sequencing has the advantage of eliminating the need for optimization of the reaction time to achieve complete deglycosylation, because the ions corresponding to the completely deglycosylated peptide can be selected for CID. However, when extensive sequence information is required, the major drawback of CID is that the generated fragments are rather specific, with preferential cleavages at X-P and D-X (X is any amino acid). In our experiments, CID-MS/MS using an ion-trap MS gave sufficient sequence information to unambiguously locate the position of the formerly glycosylated residues only for rather small peptides. Due to structural properties of mucin-type peptides (i.e., the high density of Ser, Thr, and Gly residues), the acid hydrolysis results in extensive polypeptide bond cleavages along the peptide chain. When acid hydrolysis was applied directly to the glycosylated peptide, each glycosylated hydrolytic fragment was identified by the presence of peak pairs corresponding to glycosylated and deglycosylated fragments due to the partial loss of the glycan during hydrolysis. In addition, signals corresponding to compounds resulting from the hydrolytic loss of the acetyl group from GalNAc residue were present. As a result, the spectra of the hydrolysates for the large peptides with several glycosylation sites became difficult to interpret. Transformation of the O-glycosylated Ser and Thr residues into stable methylamine derivatives was

evidenced by a concomitant mass increment of +13N× Da (N being the number of glycosylated residues) that is readily observed in the mass spectrum. The methylamine derivatives were found to be stable under acid hydrolysis, thus facilitating interpretation of the resulting spectra due to reduced sample heterogeneity. This was found especially valuable in the case of multiple glycosylated sites. Thus, a mass increment of +13N× Da on the given hydrolytic fragment unambiguously identifies the number (N) of the glycosylated sites. The drawback of this approach for its general use for site specific characterization of O-glycosylation is that the glycans are lost in the β-elimination reaction, with the consequence that information of the site-specific glycan structures is also lost. For this purpose, electron capture dissociation of the glycosylated peptides provides more comprehensive information due to its ability to cause extensive polypeptide bond fragmentation with almost no fragmentation at the glycosidic bond. Presently, however, FT-MS is not commonly available in biological laboratories. Furthermore, the sample amount requirement is higher for ECD experiments; thus, a total amount in the range of 200-400 pmol is often necessary.

In our studies of GalNAc-transferases acceptor sites’ specificity, β-elimination of O-linked glycans in a vapor of methylamine followed by partial acid hydrolysis for the determination of the formerly glycosylated residues provides an effective means for the determination of O-glycosylated site(s). Sample amount requirements are in the range of 20-50 pmol, and the instrumentation needed is commonly available in biological laboratories. ACKNOWLEDGMENT Dr. M. Hollingsworth, University of Nebraska, is acknowledged for supplying synthetic peptides; R. Koerner, for assistance with acquiring ESI mass spectra. The Danish Biotechnology Program and Danish Natural Science Council are acknowledged for financial support. This work is part of the activities of the Center for Experimental Bioinformatics sponsored by the Danish National Research Foundation. Received for review November 1, 2000. Accepted January 22, 2001. AC001288D

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