Partial Vapor-Phase Hydrolysis of Peptide Bonds: A Method for Mass Spectrometric Determination of O-Glycosylated Sites in Glycopeptides

Analytical Biochemistry 269, 54 – 65 (1999) Article ID abio.1998.3089, available online at http://www.idealibrary.com on

Partial Vapor-Phase Hydrolysis of Peptide Bonds: A Method for Mass Spectrometric Determination of O-Glycosylated Sites in Glycopeptides Ekaterina Mirgorodskaya,* Helle Hassan,† Hans H. Wandall,† Henrik Clausen,† and Peter Roepstorff * ,1 *Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark; and †School of Dentistry, Faculty of Health Science, University of Copenhagen, Nørre Alle 20, DK-2200, Copenhagen N, Denmark

Received August 11, 1998

In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. © 1999 Academic Press Key Words: glycopeptide; O-glycosylation; acid hydrolysis; mass spectrometry.

Glycosylation is one of the most common posttranslational modifications in proteins, found in nearly all biological systems ranging from primitive bacteria to mammalians and plants. The biological roles of the glycans may be only for modulation of the physiochemical properties of the protein, or their presence may 1 To whom correspondence and reprint requests should be addressed. Fax: 145 65 93 26. 61. E-mail: [email protected].

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have a critical functional role (1, 2). Therefore, determination of the glycan structures and the glycosylation sites is important for full characterization of a protein and subsequently for understanding its properties and functions. N-glycosylation always takes place on asparagine residues in the well-defined consensus sequence motifs (Asn-Xxx-Ser/Thr/Cys), Xxx Þ Pro) (3, 4). Mass spectrometric characterization of N-linked glycans using specific exo- or endoglycosidases and determination of the glycosylation sites by mass spectrometric peptide mapping are well established procedures (5–11) and have recently been reviewed (12, 13). For O-glycosylation, in contrast, no defined peptide sequence motif has been found (14) and although computer programs for prediction of potential O-glycosylation sites are available (15), such predictions are difficult and at best tentative. Determination of the factors controlling Olinked glycosylation is important, since there is evidence that alteration in O-glycosylation pattern is involved in the pathogenesis of several diseases (2). Investigation of the factors determining the sites of O-glycosylation requires assignment of the utilized sites in a large number of glycoproteins or, alternatively, characterization of the acceptor site specificities of the transferases catalyzing the addition of the first monosaccharide to the polypeptide chain. The predominant types of O-glycosylation are mucin- and yeastmannosyl-type glycosylation, which frequently occur in regions of the protein sequence with a high density of serine and threonine residues, and in the former type, often also of proline residues. In addition, O-glycosylation often protects the protein backbone against proteolysis. Therefore, proteolytic cleavage between the potential glycosylation sites prior to mass spectromet0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

MASS SPECTROMETRIC DETERMINATION OF O-GLYCOSYLATED SITES

ric peptide mapping is often not possible, especially if the potential glycosylation sites are vicinal. A number of strategies for the assessment of O-glycosylation sites have been described. The most common is complete sequencing of the glycopeptide by Edman degradation with negative identification of the glycosylated residues, but also positive identification of the glycosylated phenylthiohydantoin (PTH) 2 derivatives has been demonstrated (16). Mass spectrometric determination of O-glycosylation sites by post-source decay (PSD) analysis (17–20) and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) (21–25) has been reported. The major limitations of PSD and other MS/MS based techniques are that glycosidic bonds are more labile than polypeptide bonds and thus undergo more extensive fragmentation, resulting in low signal intensity or even absence of sequence ions still carrying the glycans. b elimination of O-linked glycans with sodium hydroxide or ammonia followed by identification by MS/MS of the resulting derivatives of formerly Oglycosylated serine and threonine residues has overcome the problem of glycosidic bond lability (26 –29). The success of MS/MS analyses is, however, highly dependent on the nature of the analyte as well as on the instrumentation. It has previously been shown that it is possible to obtain sequence information of peptides by partial acid hydrolysis followed by mass spectrometric analysis (30, 31). Tsugita et al. investigated the use of hydrolysis with different organic acids for C-terminal sequencing of peptides (32), but due to extensive internal polypeptide cleavage the acids were later replaced by acid anhydrides (33). Peptide bonds N-terminal to aspartic acid, serine, and occasionally threonine and glycine were found to be especially labile. The selective cleavages at serine and threonine are, however, particularly attractive for determination of O-glycosylation sites, since these two amino acid residues are the potential targets. In this study we demonstrate that vapor-phase acid hydrolysis combined with mass spectrometric analysis is useful for determination of O-glycosylation sites. Pentafluoropropionic acid and hydrochloric acid were used for hydrolysis of glycosylated peptides. The hydrolysis conditions were optimized to generate extensive unspecific polypeptide backbone cleavage, with minimal carbohydrate loss. Mass spectrometric analysis of the hydrolysate reveals which cleavage products carry glycans, although glycosidic bonds were partially cleaved under the conditions used. A set of observed 2

Abbreviations used: PTH, phenylthiohydantoin; PSD, postsource decay; ESI, electrospray ionization; PFPA, pentafluoropropionic acid; HCl, hydrochloric acid; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; TFA, trifluoroacetic acid.

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fragments allow mapping of the glycosylation sites. The application of acid hydrolysis for the determination of the glycosylated sites is shown for both mannosyl- and mucin-type glycosylated peptides. The examples of mucin-type glycopeptides are part of an ongoing study of the acceptor site specificities of recombinant polypeptide GalNAc-transferases (14) that require determination of glycosylated residues after in vitro glycosylation of the peptides. MATERIALS AND METHODS

Materials Pentafluoropropionic acid (PFPA) was purchased from Sigma (St. Louis, MO). Hydrochloric acid (HCl) was purchased from (Merck, Darmstadt, Germany). 2,5-Dihydroxybenzoic acid was obtained from Aldrich (Milwaukee, WI). The mannosyl-type peptide (a synthetic O-dimannosylated peptide analogue of human insulin-like growth factor 1) was a gift from M. Meldal at the Carlsberg Laboratory, Copenhagen. In Vitro Glycosylation of Peptides Synthetic mucin-derived peptides were glycosylated in vitro using recombinant polypeptide GalNAc-transferases (34, 35). GalNAc-T2 (36) was used for in vitro glycosylation of the hCG and Muc1b9 peptides, and GalNAc-T3 (37) was used for in vitro glycosylation of the Muc1a9 peptide. Acid Hydrolysis Glycosylated peptides (1–20 pmol) were lyophilized in 500-ml Eppendorf vials. The lids were removed and the vials were placed in a 22-ml glass vial with a mininert valve (Pierce, Rockford, IL). Pentafluoropropionic acid. One hundred microliters of 20% PFPA (aq), was added to the bottom of the glass vial, which was then flushed with argon. The vial was evacuated to 1 mbar, and placed in an oven at 90°C for 60 –90 min. Hydrochloric acid. One hundred microliters of 20% HCl (aq), was added to the bottom of the glass vial, which was then flushed with argon. The vial was evacuated to 1 mbar, and placed in an oven at 50°C for 90 –120 min. The hydrolysates were centrifuged in a vacuum centrifuge for 10 to 15 min to remove remaining traces of acid. Conversion of reaction intermediates into the final products was optionally accomplished by adding 10 ml 25% ammonia (aq) to the hydrolysates followed by lyophilization after 10 min incubation at room temperature.

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FIG. 1. MALDI RE–TOF mass spectra of hydrolysates of the glysosylated peptide GFYFNKPSGYGSSSRRA, which carries two mannose residues. 20 pmol of the peptide were hydrolyzed for 1 h at 90°C using (a) 90% PFPA, (b) 50% PFPA, (c) 20% PFPA, and (d) 20% PFPA after conversion of the remaining reaction intermediates by incubation with 25% NH 3 (aq). (*) and (**) Indicate an increment on the calculated peptide molecular mass corresponding to one and two mannose residues, respectively. Dots indicate stable reaction intermediates (M-18). (e) Assignment of the observed degradation products of the glycopeptide after hydrolysis with 20% PFPA. The peptide has four potential glycosylation sites, i.e., Ser 8 , Ser 12 , Ser 13 , and Ser 14 (underlined in the peptide sequence). The glycosylated peptides are detected as fully glycosylated (12Hex), partially deglycosylated (11Hex), and nonglycosylated (NG) species. The presence of molecular ions with masses corresponding to ((1– 8) 1 Hex)H 1 and ((1– 8) 1 2Hex)H 1 (indicated in bold) unambiguously identify Ser 8 as the glycosylated residue. †Calculated monoisotopic molecular masses based on the amino acid sequence. ††Measured monoisotopic masses of all peptide fragments after subtraction of the ionizing proton. The measured monoisotopic masses of the peptide fragments containing the Asn 5 residue are 1 Da higher than the calculated masses, indicating deamidation of Asn 5 during the hydrolysis.

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FIG. 1—Continued

Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Mass Spectrometry (MS) MALDI–TOF mass spectra were acquired in linear (lin) and reflector (ref) mode on a Voyager-Elite Biospectrometry Workstation (PerSeptive Biosystems Inc., Framingham, MA) equipped with delayed extraction technology. Mass spectra were externally calibrated unless otherwise stated. The hydrolyzed samples were dissolved in 0.1% trifluoroacetic acid (TFA) to a concentration of 1– 4 pmol/ml, based on the initial amount of peptide subjected to hydrolysis, and prepared for MALDI–MS analysis by mixing 1 ml of sample solution with 1 ml of matrix solution (2,5-dihydroxybenzoic acid, 10 mg/ml in acetonitrile: 0.1% TFA, 1:2 (v/v)) directly on the target. ESI–Mass Spectrometry ESI mass spectra were acquired on an Esquire Ion Trap mass spectrometer (Bruker-Franzen Analytik GmbH, Bremen, Germany), equipped with a nanoelectrospray source. One hundred to 150 scans were summed. Hydrolyzed samples were dissolved in water: methanol, 1:1 (v/v) to a final concentration of 5 pmol/ml, based on the initial amount of peptide subjected to hydrolysis, whereof 2 ml were used for ESI–MS analysis. Data Interpretation The protein analysis software GPMAW (htpp://www. welcome.to/gpmaw; Lighthouse Data, Odense, Den-

mark) was used for data interpretation. The observed peptide masses were searched against the theoretically calculated masses for all possible hydrolytic peptides without as well as with mass increments corresponding to attached monosaccharide residues. RESULTS

1. Determination of Optimal Hydrolysis Conditions Unambiguous identification of glycosylation sites requires cleavages between all potential sites. Therefore we optimized the hydrolysis conditions for the generation of extensive sequence ladders by unspecific inchain cleavage with a minimal degree of O-glycosidic bond cleavage. The effects of the PFPA concentration and the hydrolysis time were studied using the synthetic glycosylated peptide GFYFNKPSGYGSSSRRA with known glycan attachment site as model substance. The peptide has four potential glycosylation sites; one on Ser 8 and a cluster of three vicinal sites on Ser 12, Ser 13, and Ser 14, and carries two mannose residues attached to Ser 8. Initial experiments showed that the glycan was only partially released during vapor-phase hydrolysis with PFPA. Consequently, the glycosylated peptides generated by hydrolysis are detected as molecular ions corresponding to the fully glycosylated, partially deglycosylated, and completely deglycosylated peptides. Hydrolysis with varying PFPA concentrations (10 – 90%) was performed to evaluate the optimal concentra-

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FIG. 2. (a) MALDI–TOF mass spectrum of the glycosylated peptide Muc1b9 (RPAPGSTAPPA) after hydrolysis with 20% PFPA at 90°C for 1 h. Partial loss of the GalNAc residue is observed for all glycosylated hydrolytic fragments. (*) Indicates loss of the acetyl group (242 Da) from the GalNAc residue, observed for all glycosylated hydrolytic fragments. (b) The observed degradation products of the peptide Muc1b9. The peptide has two vicinal potential glycosylation sites (underlined in the peptide sequence), i.e., Ser 6, Thr 7. The glycosylated peptide fragments are detected as nonglycosylated (NG) and glycosylated (11GalNAc) product ions, accompanied by loss of the acetyl group from the GalNAc residue (masses given in parentheses). The hydrolytic peptide fragments essential for the determination of the glycosylation site as Thr 7 are indicated in boldface. †Calculated monoisotopic molecular masses based on the amino acid sequence. ††Measured monoisotopic masses of all peptide fragments after subtraction of the ionizing proton.

tion for identification of the glycosylated sites. Figure 1 shows the MALDI–TOF mass spectra of the products obtained from 1-h hydrolysis of the model peptide using (a) 90%, (b) 50%, and (c) 20% PFPA. Ninety percent PFPA resulted primarily in specific cleavages at the N-terminal side of Ser as reported by Tsugita et al. (32), as well as in partial cleavage of the glycosidic bond (Fig. 1a). The most abundant hydrolysis product in the spectrum is peptide fragment (1–11), indicating that the cleavages N-terminal to the serine residues were too rapid, resulting in low intensities of the peaks corresponding to the fragments (1–12 and 1–13). No cleavage was observed at the N-terminal side of Ser 8, in agreement with the previously reported stability of

bonds C-terminal to Pro residues (32). PFPA concentrations between 70 and 40% yielded more extensive cleavages of both the polypeptide backbone and the glycosidic bond as illustrated with the MALDI–TOF mass spectrum of the products from 1-h hydrolysis with 50% PFPA (Fig. 1b). Further decrease of the PFPA concentration to 20% resulted in considerable decrease of glycosidic bond cleavage, but with retained efficient polypeptide backbone cleavages (Fig. 1c). The hydrolysis of the peptide with 20% PFPA at 90°C for 1 h resulted in generation of N- and C-terminal sequence ladders, which allowed distinction between all the potential glycosylation sites and unambiguously identified Ser 8 to be the glycosylated residue (Fig. 1e).

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FIG. 3. (a) ESI ion trap mass spectrum of the glycosylated peptide Muc1a9 (AHGVTSAPDTR) after hydrolysis with 20% PFPA at 90°C for 1.5 h. (*) indicates loss of the acetyl group (242 Da) from the GalNAc residue. (b) The observed degradation products of the peptide Muc1a9. The peptide has three potential glycosylation sites, i.e., Thr 5, Ser 6, and Thr 10 (underlined in the peptide sequence). The glycosylated peptide fragments are detected as nonglycosylated (NG) and glycosylated (11GalNAc) product ions, accompanied by loss of the acetyl group from the GalNAc residue (masses given in parentheses). The presence of a GalNAc residue on the peptide (1–5), indicated in boldface, unambiguously identifies Thr 5 as the glycosylated residue. †Calculated monoisotopic molecular masses based on the amino acid sequence. ††Measured monoisotopic masses of all peptide fragments after subtraction of the ionizing proton.

PFPA concentrations lower than 20% resulted in insufficient polypeptide backbone cleavage. Prolongation of the hydrolysis time resulted in increased hydrolysis of both peptide and O-glycosidic bonds with no preference for either type. Although it was possible to assign the glycosylation site for the given peptide from a variety of

concentration and time combinations, hydrolysis with 20% PFPA for 1 h was found to be optimal for efficient generation of polypeptide ladders with minimal carbohydrate loss. Deamidation of the asparagine residue was observed for all PFPA concentrations and seems to be unavoidable.

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FIG. 4. MALDI RE–TOF mass spectra of the glycosylated peptide Muc1a9 (AHGVTSAPDTR) after hydrolysis with (a) 20% PFPA at 90°C for 1 h and (b) 20% HCl at 50°C for 1.5 h. (*) Indicates loss of the acetyl group (242 Da) from the GalNAc residue.

In all experiments, most of the observed molecular ions corresponding to the products of hydrolysis were accompanied by additional signals of M-18 Da and M-1 Da. The M-18 Da products (marked with dots in Figs. 1a–1c) correspond to oxazolone intermediates and have previously been reported (32). For complex hydrolysates, the presence of such products may result in ambiguous signal assignment. Thus, for example, the signal of m/z 1486.81 assigned as (1–12)* in Figs. 1a–1c could also be assigned as the reaction intermediate ((1–10)**-18 Da) (calculated monoisotopic molec-

ular masses of these hydrolytic products, taking into account deamidation of Asn, are 1485.63 and 1485.62 Da, respectively). Tsugita et al. (38) exposed the hydrolysates to aqueous pyridine vapor for 10 min at 100°C for efficient conversion of the remaining reaction intermediates into the final products. We found that these intermediates could be quantitatively converted to the final products by incubation with 25% ammonia solution at room temperature for 10 min (Fig. 1d). The above-mentioned ambiguity between the (1–12)* or the (1–10)** oxazolone intermediate was resolved after

MASS SPECTROMETRIC DETERMINATION OF O-GLYCOSYLATED SITES

this additional step. The presence of products with M-1 Da has been previously observed for hydrolysis with perfluoroacyl anhydride vapor and has been ascribed to cleavage between the amido group and the a-carbon of the adjacent amino acid residue, resulting in formation of a cleavage product with amidated C-terminus (38). We observed that the M-1 Da signals were less abundant when the PFPA concentration was decreased. The presence of M-1 Da species may also lead to signal interpretation ambiguities; i.e., its observation can be due to either amidated C-terminus or incomplete deamidation of Asn or Gln in the cleavage products. Comparison of the signals corresponding to the M-18 Da oxazolone intermediates and the final products showed that the M-1 Da signals were only observed for the final products, and not for the reaction intermediates. This demonstrated that the deamidation of the Asn residue was complete and supported the previous report that the M-1 Da signals are due to partially amidated Cterminus of the hydrolytic fragments. 2. Mucin-Type Glycosylation The main purpose of this study was the development of a procedure for the determination of glycosylated sites in mucin type peptides after in vitro glycosylation with different recombinant polypeptide GalNAc-transferases (GalNAc-T1, -T2, -T3). Two in vitro glycosylated mucin-derived peptides, Muc1a9 (AHGVTSAPDTR) and Muc1b9 (RPAPGSTAPPA), the glycosylation sites of which were previously determined using Edman sequencing (35), were used to investigate the performance of the method for identification of mucin-type glycosylation sites. The peptide Muc1b9 has two vicinal potential glycosylation sites. The difference between the observed (1223.7 Da) and the calculated (1020.5 Da) monoisotopic molecular masses of the peptide after in vitro glycosylation with GalNAc-T2 indicates the incorporation of one N-acetylgalactosamine (GalNAc) residue. To determine which of the two sites was utilized, 20 pmol of the peptide was hydrolyzed with 20% PFPA at 90°C for 1 h and an aliquot of the hydrolysate was analyzed by MALDI–MS (Fig. 2a). Peptide fragments containing the utilized glycosylation site were detected with masses corresponding to both glycosylated and deglycosylated species, due to partial hydrolysis of the glycosidic bond, whereas peptide fragments not containing the utilized glycosylation site were only detected with masses corresponding to the nonglycosylated species. In addition, loss of the acetyl group (242 Da) from the GalNAc residue was observed for all glycosylated hydrolytic peptide fragments. The glycosylated site could be assigned due to the detection of a mass increment corresponding to one GalNAc residue

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on peptide (6 –11), but not on peptide (1– 6), thus, identifying Thr 7 as the glycosylated residue (Fig. 2b). The same glycosylated site was observed for the peptide glycosylated with GalNAc-T1 and -T3 transferases (data not shown), showing overlapping specificities of the three transferases toward this peptide. These results are in agreement with previous studies using other techniques (17, 35). Peptide Muc1a9 has three potential glycosylation sites. Based on the difference between the observed (1313.3 Da) and the calculated (1110.5 Da) monoisotopic molecular masses, the GalNAc-T3 in vitro glycosylated peptide was assumed to carry one GalNAc residue. To determine the utilized glycosylation site, 20 pmol of the peptide was hydrolyzed with 20% PFPA at 90°C for 1.5 h. The hydrolysate was analyzed directly by nano-ESI–MS without any purification prior to analysis (Fig. 3a). Assignment of the observed degradation products is shown in Fig. 3b. The observed Nand C-terminal sequence ladders unambiguously localize the glycosylation site to Thr 5 (Fig. 3b). The same glycosylated site was observed for the peptide glycosylated with GalNAc-T1 and -T2 transferases (data not shown). These results are in agreement with the previously reported acceptor site for GalNAc-T1, -T2, and -T3 transferases (35). Formation of the intermediate products at M-18 Da was also observed for these two peptides, but to a much lesser extent than for the mannosyl-type glycosylated model peptide. M-1 Da products were not observed at all in the spectra of the mucin-type peptides. For both peptides, partial loss of the acetyl groups (242 Da) from the GalNAc residues was observed for all glycosylated hydrolytic fragments (Figs. 2a and 3a). The partial loss of the acetyl group increases the complexity of the mass spectra and decreases the intensity of the peaks corresponding to glycosylated peptide fragments. Therefore, whether altered hydrolysis conditions could reduce the loss of the acetyl group was investigated. It was found that PFPA hydrolysis always resulted in acetyl loss, whereas hydrolysis with vapor of HCl at 50°C for 1.5 h yielded similar hydrolytic fragments, but with significantly reduced loss of the acetyl group (Fig. 4). As an example of a peptide with several utilized glycosylation sites, the in vitro glycosylated peptide hCG (PRFQDSSSSKAPPPSLPSPSRLPG) was studied. Analysis of the acceptor substrate specificity of three recombinant polypeptide GalNAc-transferases (T1, T2, and T3) has shown that only GalNAc-T2 catalyzes transfer of GalNAc residues to this peptide sequence (35, 36). MALDI–MS analysis of the in vitro terminally glycosylated peptide gave a mass difference of 609.22 Da between measured and calculated monoisotopic molecular masses, indicating incorporation

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FIG. 5. MALDI RE–TOF mass spectra of the glycosylated peptide hCG (PRFQDSSSSKAPPPSLPSPSRLPG) after hydrolysis with (a) 20% PFPA at 90°C for 1 h and (b) 20% HCl at 50°C for 1.5 h. Only the signals essential for determination of glycosylation sites are labeled in the spectrum. (*) Indicates loss of acetyl groups (242 Da) from GalNAc residues. (¤) Indicates unidentified ion signals. (c) Assignment of all observed degradation products of the glycopeptide hCG after hydrolysis with 20% PFPA. The peptide has seven potential glycosylation sites, i.e., a cluster of four vicinal sites (Ser 6, Ser 7, Ser 8, and Ser 9) and three separated sites (Ser 15, Ser 18, and Ser 20), underlined in the peptide sequence. The glycosylated peptides are detected as fully glycosylated (13GalNAc), partially deglycosylated (12GalNAc, 11GalNAc), and nonglycosylated (NG) product ions. Masses in parentheses correspond to partial loss of acetyl groups from GalNAc residues. The hydrolytic peptide fragments essential for determination of the glycosylation sites (indicated in boldface) identify Ser 9, Ser 15, and Ser 20 as the glycosylated residues. †Calculated monoisotopic molecular masses based on the amino acid sequence. ††Measured monoisotopic masses of all peptide fragments after subtraction of the ionizing proton. The observed masses of the peptide fragments containing the Gln 4 residue are 1 Da higher than the calculated masses, indicating deamidation of Gln 4 during the hydrolysis. Hydrolytic fragments also observed when 20% HCl was used for hydrolysis of the peptide are indicated by “h.”

of three GalNAc residues. The peptide has seven potential glycosylation sites: a cluster of four vicinal sites (Ser 6, Ser 7, Ser 8, and Ser 9) and three additional sites (Ser 15, Ser 18, and Ser 20). The attempt to determine the glycosylated residues by direct Edman sequencing of the peptide did not allow identification of the attachment sites, mainly due to strong signal degradation already after 11 cycles (data not shown). The peptide was subjected to acid hydrolysis using both PFPA and HCl, and the resulting mixtures were analyzed by MALDI–MS (Figs. 5a and 5b).

Hydrolysis of the glycosylated peptide with 20% PFPA at 90°C for 1 h generated a highly complex mixture: more than 150 peptide signals were detected by MALDI–TOF–MS analysis (Fig. 5a). In spite of the complexity of the peptide mixture, it was possible to assign almost all well-resolved peaks in the spectrum except for a few peaks mainly in the low-mass region (labeled with ¤ in Fig. 5a). The MALDI–TOF mass spectrum contains several series of peaks, most of which belong to a C-terminal sequence ladder and accompanying partial losses of one to three GalNAc res-

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FIG. 5—Continued

idues and acetyl groups. Their observed monoisotopic masses after subtraction of the ionizing proton are listed in Fig. 5c. To resolve ambiguities in the signal interpretation, the externally calibrated spectrum was internally recalibrated using the calculated monoisotopic masses of a few unambiguously assigned peaks, namely peptide fragments 17–24 (809.44 Da) and 6 –24 (1847.97 Da). The resulting improved mass accuracy (better than 0.1 Da over the entire mass range) resolved several assignment ambiguities. The retrieved peptide mass list was searched against theoretically calculated peptide fragment masses, including mass increments corresponding to monosaccharide residues, using the protein analysis software GPMAW. Signal assignment revealed the presence of an extensive Cterminal sequence ladder and a short N-terminal sequence ladder (Fig. 5c). In addition, several peaks were assigned as internal hydrolytic fragments following favored acid hydrolytic cleavage sites, i.e., the Cterminal side of the aspartic acid, the N-terminal of serine, and both sides of glycine (39). However, these internal fragments were of low abundance and represented only a minor part of the detected molecular ions and are therefore not listed in Fig. 5c. The observed C-terminal sequence ladder was sufficient to assign the glycosylated sites as Ser 9, Ser 15, and Ser 20 (Fig. 5c), which is in agreement with a previous report for in vivo

glycosylated human chorionic gonadotropin (40) of which the studied peptide is a partial sequence. The MALDI–TOF mass spectrum of the mixture derived by HCl hydrolysis of the glycosylated peptide with 20% HCl at 50°C for 1.5 h is shown in Fig. 5b. HCl hydrolysis of the peptide generated products similar to those obtained after PFPA hydrolysis, i.e., mainly a C-terminal sequence ladder with accompanying losses of GalNAc residues and acetyl groups (indicated by “h” in Fig. 5c). In this case, the detected degradation products also allowed identification of the glycosylated residues. The mass spectrum acquired after HCl hydrolysis was considerably less complex compared to the spectrum obtained after PFPA hydrolysis, mainly due to reduced loss of acetyl groups. On the other hand, the partial loss of acetyl groups was found to be useful for signal interpretation of the spectrum, since the presence of 242 Da species is indicative of glycosylated peptide fragments. In addition, their signal intensities relative to their acetylated counterparts strongly support the assignment of the number of GalNAc residues in a given hydrolytic product ion. To confirm the assignment of the glycosylated sites, the peptide was subjected to proteolysis with endoproteinase Lys-C followed by HPLC separation of the resulting two peptides. Each of these was analyzed by MALDI–MS before and after PFPA hydrolysis. The

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MALDI mass spectra obtained prior to hydrolysis confirmed that the proteolytic peptide fragment (1–10) carried only one GalNAc residue, whereas the proteolytic peptide fragment (11–24) carried two. Interpretation of the spectra after acid hydrolysis of the Lys-C generated peptides was considerably simpler than that of the entire peptide and confirmed the previous assignment (spectra not shown). DISCUSSION

The original purpose of this study was to develop a method that would allow determination of the glycosylated sites in mucin-derived peptides, in vitro glycosylated with different recombinant polypeptide GalNActransferases to characterize their activities. The application of acid hydrolysis for the determination of O-glycosylation sites requires extensive peptide bond cleavages to distinguish between all potential sites, concomitantly with minimal hydrolysis of the glycosidic bonds. Hydrolysis in vapor of 20% PFPA at 90°C for 1–1.5 h or 20% HCl at 50°C for 1.5–2 h was found optimal to fulfill the above-mentioned requirements. The hydrolysis mainly results in generation of Nand/or C-terminal sequence ladders allowing discrimination between potential glycosylation sites. Due to the partial cleavage of glycosidic bonds, glycosylated peptides generated by hydrolysis are detected as molecular ions corresponding to fully glycosylated, partially deglycosylated, and completely deglycosylated peptides. The peptide mixtures generated by PFPA hydrolysis reactions are often highly complex, especially if the peptides contain several utilized glycosylation sites; i.e., the mixture contains intermediate reaction products as well as products generated by partial loss of the glycans. For mucin-type glycopeptides, partial loss of the acetyl group from GalNAc residues further increases the heterogeneity of the hydrolysates. To reduce complexity of the spectra, the remaining reaction intermediates (M-18 Da) can be converted into the final products by a brief incubation with 25% ammonia (aq). The losses of acetyl groups, in the case of the mucintype glycopeptide, can be minimized by using HCl instead of PFPA. This reduces the sample heterogeneity, but also leads to loss of useful information, since the presence and intensity of peaks corresponding to these losses have a considerable diagnostic value; i.e., the presence of ions 42 Da less than the MH 1 ion confirms that the molecular ion corresponds to the glycosylated form of the peptide. Therefore, PFPA is often a better choice than HCl. Since the amino acid sequence of the peptides is normally known, the masses of all potential hydrolysis products can be calculated and molecular ions for the nonglycosylated peptides can be identified

in the spectra. The presence of an ion 203 Da above a calculated MH 1 for a nonglycosylated peptide indicates the presence of a GalNAc residue on the given peptide. The same strategy can be applied to mannosyl-type glycosylation, resulting in a mass increment of 162 Da. In general, the interpretation strategy implies knowledge of the peptide sequence. This, however, will normally be the case in the search for the specific position of posttranslational modifications such as O-glycosylation. Due to the partial loss of the glycans during hydrolysis, the method does not allow the distinction between a partially and a fully glycosylated sites. Such a distinction may be crucial for studies of the regulatory role of certain types of O-glycosylation. For this purpose, further optimization or refinement of the described procedure is needed. Vapor-phase PFPA hydrolysis in combination with mass spectrometric analysis proved rapid and efficient for determination of the O-glycosylated sites in both mucin- and mannosyl-type glycopeptides. The experimental setup is inexpensive and simple, and the method is compatible with most types of mass spectrometers. The advantage of performing microscale derivatization of peptides and glycoconjugates in the vapor phase followed by mass spectrometric analysis has previously been reported (41, 42). Performing the hydrolysis in the vapor phase eliminates the risk of contaminating the sample with impurities from reagents and solvents, thus allowing analysis of the reaction products, both by MALDI–MS and ESI–MS, without the need for further purification. Although MALDI–MS is often favored for analysis of complex mixtures, it has a limitation for detection of low-molecular-mass hydrolytic fragments due to interference with matrix-derived signals. For the detection of such low-mass fragments ESI–MS is favored despite its lower sensitivity. The mechanisms involved in acid hydrolysis of polypeptides have not yet been fully elucidated. For example, although formation of the M-1 species has previously been ascribed to cleavage between the amido group and the a-carbon of the adjacent amino acid residue instead of peptide bond, resulting in formation of a Cterminal amide (38), the mechanism for the formation of these products is not known. It is, however, our observation that the M-1 Da signals are mainly observed for ladders generated by C-terminal degradation of the peptide. The factors directing specific or random bond cleavage also need further elucidation to effectively exploit the vapor-phase hydrolysis method that is highly compatible with subsequent mass spectrometric analysis. Studies for alternative exploitation of partial acid hydrolysis and for developing procedures, which also allow assignment of partial glycosylation, are presently underway in our laboratory.

MASS SPECTROMETRIC DETERMINATION OF O-GLYCOSYLATED SITES

ACKNOWLEDGMENTS Dr. M. Hollingsworth, University of Nebraska, is acknowledged for supplying synthetic peptides Muc1a9 and Muc1b9, Dr. B. Svensson is acknowledged for supplying the mannosyl-type glycosylated peptide, R. Koerner is acknowledged for assistance with acquiring ESI mass spectra, and Dr. E. Nordhoff and J. Gobom are thanked for valuable discussions. The Danish Biotechnology Program and Danish Natural Science Council are acknowledged for financial support.

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