Mass Spectrometric Determination of the Sites of O-Glycan Attachment with Low Picomolar Sensitivity

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

257, 149 –160 (1998)

AB972548

Mass Spectrometric Determination of the Sites of O-Glycan Attachment with Low Picomolar Sensitivity Geert Jan Rademaker,*,1 Spiros A. Pergantis,*,2 Leonore Blok-Tip,* James I. Langridge,† Astrid Kleen,‡,3 and Jane E. Thomas-Oates*,4 *Department of Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; †Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom; and ‡Institut fu¨r Organische Chemie, Universita¨t Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany

Received July 28, 1997

A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on b-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandem mass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material. © 1998 Academic Press

Of all possible forms of posttranslational modification, O-glycosylation is possibly the most challenging to the structural chemist, since the definition of the structure of an O-glycopeptide involves not only the determination of the structure of the glycan moiety, but also requires that the site of attachment of the glycan to the peptide chain be defined. In spite of many efforts, to date, no consensus sequence for O-glycosylation has been described (1– 6). The assignment of the 1 Present address: Glycoprotein Structure/Function Group, Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK. 2 Present address: Department of Chemistry, Birkbeck College, Gordon House, 29, Gordon Square, London WC1H 0PP, UK. 3 Present address: Henkel KGaA, Henkelstrasse 67, 40191 Du¨sseldorf, Germany. 4 To whom correspondence should be addressed. Fax: 131.30.2518219. E-mail: [email protected].

0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

site of attachment of the glycan is further complicated by the fact that there may be many sites of O-glycan attachment adjacent to each other and that these are frequently located in a region of peptide sequence in which many serine and threonine residues are found together, all of which may or may not be glycosylated (3). During Edman sequencing, glycosylated amino acid residues are not recovered, as they do not coelute with amino acid standards, and thus yield a ‘‘blank’’ which has been used as an indication for a glycosylated residue (7). The failure to identify a consensus sequence for O-glycosylation, together with the fact that no pan-specific enzyme for the removal of O-glycans has been reported, means that identifying the site of O-glycosylation is much more complicated than for Nglycosylation. Although mass spectrometry of peptides is well suited to the rapid and sensitive sequencing of peptides (8, 9), mass spectrometric analysis of intact glycopeptides is rather disappointing; the most frequently observed fragmentation is that corresponding to the loss of the glycan, leaving the site of attachment unmodified. In favorable cases, however, some fragmentation of the peptide backbone with retention of the glycan is observed, allowing the site to be identified (10 –12). However, this seems to be very much dependent on the structure of the peptide and on the experimental conditions used and is therefore unsuitable for incorporation into a generally applicable protocol. We have shown that the use of b-elimination can yield valuable information on the site of base-labile serine- or threonine-bound O-glycans(13). Unfortunately, it has become apparent to us that the use of reductant during b-elimination to prevent peeling of the released O-glycan (14) can degrade the peptide backbone of certain peptides (15, 16). The reason for 149

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this vulnerability, and the influence of the nature of the peptide backbone on its stability under these conditions, is unfortunately not at all clear. The particular vulnerability of the -Thr-Pro- sequence reported by other workers (15) seems to have been more dependent on the rest of the peptide backbone than was assumed at that time (16). Modification of the protocol, omitting the reductant, circumvents the problem of peptide degradation (16, 17). Although the usefulness of b-elimination in the analysis of serine- or threonine-bound O-glycopeptides has clearly been shown, developments in instrumentation in the meantime mean that the sensitivity then reported as being good now leaves much to be desired. In our original paper fast atom bombardment collisioninduced dissociation tandem mass spectrometric (FAB5 CID MS–MS) analysis was used and 5 nmol of starting material was required (13), while a later report described a similar protocol based on electrospray ionization collision-induced dissociation tandem mass spectrometric (ESI CID MS–MS) analysis and while claiming much improved sensitivity still required close to 1 nmol of starting material (18). In this paper we describe the development and optimization of a protocol for the identification of O-glycosylation sites using a much more sensitive procedure that also has the added benefits of even simpler sample handling and in which only low picomolar amounts of starting material are required, making the analysis more widely applicable to the amounts of sample usually available from biological sources. MATERALS AND METHODS

Peptides and Glycopeptides Biologically active peptide and glycopeptide compounds 1–11 (see below) were prepared simultaneously using multiple-column solid-phase synthesis using a custom-made 20-well peptide synthesizer (19–21), following the azide strategy (22, 23). The synthesis was performed on a kieselguhr-supported poly(dimethylacrylamide) resin (24, 25) as the solid support, which was derivatized with norleucine as an internal reference amino acid and a peptide amide linker [RINK-linker (26)]. Suitable protected nonglycosylated and glycosylated Na-fluoren-9-ylmethoxycarbonyl amino acid pentafluorophenyl esters [Fmoc-amino acid-OPfp (27, 28)] were used as building blocks and coupled stepwise starting at the C-terminus and continuing toward the N-terminus by application of the respective activated amino acids. The addition of equimolar amounts of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3benzotriazine as an auxiliary nucleophile both allowed 5 Abbreviations used: CID, collision-induced dissociation; ESI, electrospray ionization; FAB, fast atom bombardment; MS–MS, tandem mass spectrometry.

the progress of the peptide-bond formation to be monitored visually and enhanced the reactivity of the Pfpester (29). After coupling of the last amino acid, the Fmoc group was removed and the terminal amino group was acetylated. Glycosylated amino acids were prepared by stereoselective a-glycosidic linkage of Na-Fmoc-Thr-OPfp and Na-Fmoc-Ser-OPfp to the peracetylated glycosyl halides of D-GalN3 p or b-Galp-(1 3 3)-D-GalN3 p. The resulting azido glycosyl amino acids were introduced directly into the peptide chain by solid-phase synthesis (22, 23). When peptide assembly was complete, transformation of the azido group of the glycosyl amino acid into the desired acetamido derivative was performed by treating the resin-bound glycopeptide with thioacetic acid. The progress of the reaction was monitored spectroscopically by following the disappearance of the azido IR absorption band.

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brated with 2 ml 5% aqueous acetic acid. The sample was applied to the column, which was then washed with 2 ml 5% aqueous acetic acid, after which the released peptide was eluted with 1 ml 20% propan-1-ol (p.a.; Merck) in 5% acetic acid and 1 ml 40% propan1-ol in 5% acetic acid. The propanol-containing fractions were pooled and dried using a vacuum centrifuge and stored at 220°C prior to analysis. Desalting was alternatively carried out using Microcon-SCX cartridges (Amicon Inc., Beverly, MA) (Scheme 1, b2) according to the manufacturer’s specifications for maximum recovery, except that washing with 500 ml 10 mM HCl in distilled water was carried out twice, instead of the recommended once in order to remove all residual salt. Sample Handling for Q-Tof Experiment

SCHEME 1. Comparison of protocols for release of peptide from O-linked glycopeptide using b-elimination.

Cleavage of the peptide or glycopeptides from the resin by treatment with trifluoroacetic acid followed by de-O-acetylation of the glycan moiety and RPHPLC purification yielded peptide 1 in 85% yield and glycopeptides 2–11 in 32– 47% overall yield (calculation based on the substitution of the resin). The purity of all compounds, determined using analytical HPLC, 1H NMR spectroscopy, and FAB–MS, was excellent (20, 30, 31). The compounds were then used to investigate the substrate specificity of glycosyltransferases (32, 33).

b-Elimination Using NH4OH. The peptide or glycopeptide (10 – 1000 pmol) in a plastic Micro tube (Sarstedt, Germany) was dissolved in 300 ml 25% NH4OH (p.a.; Merck, Darmstadt, Germany) and incubated at 45°C for 0 –36 h (Scheme 1). The reaction was subsequently stopped by removing the reagent under reduced pressure (vacuum centrifuge). The sample was stored at 220°C prior to analysis and used without further purification. Using NaOH. To the glycopeptide (100 or 500 pmol) in a 1.5-ml plastic Micro tube, 100 ml of 0.1 M NaOH (p.a.; Lamers & Pleuger BV, ’s-Hertogenbosch, The Netherlands) was added. The mixture was incubated at 45°C for 4 h and subsequently quenched by adjusting the pH to 4 –5 with glacial acetic acid (p.a.; Merck). The mixture was desalted (Scheme 1, b1) using a C18 solid-phase extraction column (50 mg of sorbent) (Alltech Nederland BV, Breda, The Netherlands), preconditioned with 1 ml methanol (HPLC-grade; Biosolve Ltd., Valkenswaard, The Netherlands), and equili-

To 500 ml of 25% NH4OH (Suprapur, Merck) in sterile BioStor vials (National Scientific Supply Co. Inc., Claremont, USA) 10, 5, or 1 pmol of glycopeptide was added. The mixture was incubated at 45°C for 8 h, after which the reagent was removed by vacuum centrifugation. The samples were then redissolved in 200 ml of 50% aqueous methanol, dried again, and stored at 220°C. The samples were analyzed without further purification. Mass Spectrometric Conditions ESI mass spectra were acquired using two different types of mass spectrometers. Initially, an ESI singlequadrupole mass spectrometer (VG Platform II; Micromass, Wythenshawe, Manchester, UK) was used to monitor the products resulting from the b-elimination reaction of the different glycopeptides studied. Analysis of the reaction mixture was conducted in the flow injection mode. A Shimadzu LC9A HPLC pump was used to deliver an aqueous carrier containing 50% acetonitrile and 0.1% formic acid at a flow rate of 40 ml/min. A Rheodyne 2698 injector fitted with a 10-ml loop was used for sample introduction. Prior to the analysis of the reaction mixtures, optimization of the ESI response for the [M 1 Na]1 pseudomolecular ion corresponding to peptide 8 was accomplished by injecting approximately 100 ml of a 0.1 nmol/ml solution of peptide 8. This solution was sufficient to provide quasicontinuous sample introduction and thus allows the apparent optimal response to be reached by iteratively varying all instrument parameters. The incubation reaction products were redissolved in 25 ml eluent, 10 ml of which was injected, unless otherwise stated. All spectra were recorded in the positive-ion ESI mode. Following optimization the sample cone voltage was set at ;88 V, the capillary voltage at ; 3.75 kV, and the source temperature at 80°C. The scan range was

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from 600 to 1200 amu (scan speed 4 s), unless otherwise stated. Mass spectra were recorded in the continuum mode, subtracted, and smoothed once using Masslynx software (Micromass). Q-Tof Conditions ESI mass spectra (survey scans) and CID tandem MS data were acquired on a Micromass Q-Tof mass spectrometer (Wythenshawe) fitted with a Nanoflow electrospray ion source. Peptide samples were dissolved in 50/50 (v/v) methanol/0.2% formic acid, and 1-ml aliquots of these solutions were loaded into 1-mm borosilicate Nanoflow tips. The mass spectrometer was operated in the positiveion mode with a source temperature of 30°C and a drying gas flow rate of 40 liters/h. A potential of 0.8 – 1.5 kV applied to the Nanoflow tip combined with a nitrogen backpressure of 5–10 psi produced a sample flow rate of 10 –30 nl/min into the analyzer. All data, both MS and CID tandem MS, were acquired with the Tof analyzer and data integrated every 10 s. In MS mode the quadrupole was used in Rf-only mode and transmitted about a decade in mass to the Tof. In tandem mode the quadrupole was used in resolving mode to select the precursor ion for fragmentation in the hexapole collision cell. CID tandem MS was performed with argon gas in the collision cell at a pressure of 6 3 1025 mbar measured in the analyzer. A collision energy of 50 eV was used for the peptide. RESULTS AND DISCUSSION

To release the glycan from an O-linked glycopeptide using b-elimination, NaOH is commonly used. When the released glycans are to be studied, the use of reductant is required to avoid peeling (14). However, the reductant can, under some circumstances, degrade the peptide, although it is as yet unclear why some peptides undergo this degradation, whereas others appear to be totally unaffected (15, 16). Consequently we have recommended that the method we published for release of peptides from O-linked glycopeptides (13) can be modified by the omission of the reductant (16, 17). However, time-course studies of peptide release using NaOH and using ESI–MS monitoring (data not shown) show that although the peptide is released efficiently, some peptides may then be rapidly degraded on longer incubation. In addition, we have observed that the use of NaOH as the b-elimination reagent is not ideal when the peptides released are to be sequenced by studying the collisionally induced fragmentation of the protonated peptide, especially if the peptide is ionized using ESI. The introduction of large quantities of sodium makes a rigorous sample clean-up necessary, which unavoidably results in significant loss of material. Fur-

thermore, we have observed that a C-terminal amide is labile under the conditions of b-elimination using NaOH (13, 16). In an attempt to circumvent the problems of sodium contamination and peptide degradation, and hoping to increase overall sensitivity by removing the need for most of the sample-handling steps, we have examined the feasibility of using NH4OH in place of NaOH as the b-elimination reagent. The reagent’s volatility clearly has the advantage of facilitating its removal, thus obviating the need for desalting. Because NH4OH is a weaker base than NaOH, a higher concentration is required; a commercially available 25% solution with a pH only slightly lower than that of 0.1 M aqueous NaOH was used for the experiments described in this paper. Termination of the reaction was achieved simply by removing the reagent under reduced pressure. The effects of NH4OH treatment on a nonglycosylated peptide of comparable sequence to that of the glycopeptides available were determined using peptide 1. The ESI mass spectrum recorded from peptide 1 (Fig. 1A) obtained after the peptide had been subjected to NH4OH treatment (4 h) contains a base peak at m/z 852 which corresponds to the [M 1 Na]1 pseudomolecular ion for the peptide. An ion of identical m/z value was observed after analyzing untreated peptide 1 (Fig. 1B). This is in marked contrast with the data obtained from peptide 1 after NaOH treatment, when a mass increase of 1 amu is observed, corresponding to the conversion of the terminal amide to the free acid (13, 16). Since the peptide is thus clearly stable to treatment with NH4OH, we were encouraged to study the behavior of glycopeptides under similar conditions. When glycopeptides 2 and 3 were subjected to b-elimination using NH4OH for 1, 2, 4, 6, and 8 h, the ESI mass spectra recorded contained [M 1 Na]1 pseudomolecular ions corresponding to the unreleased glycopeptides (m/z 1055 and 1041, respectively). The relative intensity of these ions declines as the incubation time increases, and in the spectra obtained from compound 2 the ion at m/z 1055 is much lower after 8 h reaction time than after only 4 h (Figs. 2A and 2B). In the spectra from both compounds, the base peak (m/z 851 from compound 2 and m/z 837 from compound 3) corresponds not to an [M 1 Na]1 pseudomolecular ion for the released peptide (rPep), but instead to [rPep 1 17 1 Na]1. The appearance of this pseudomolecular ion clearly demonstrates that the glycan has been eliminated. The signal corresponding to [rPep 1 17 1 Na]1 was observed in the spectra from all the glycopeptides examined following b-elimination using NH4OH. The mass difference of 17 amu was investigated in detail using high-energy FAB CID MS–MS analyses of the product from compound 2. The tandem mass spectrum (Fig. 2C) contains an almost complete series of A, B, and Y0 ions that clearly shows that the 17-amu incre-

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FIG. 1. Effect of NH4OH treatment on peptide 1. (A) ESI mass spectrum obtained on incubating 1 nmol for 4 h in NH4OH. (B) ESI mass spectrum obtained under similar conditions after 0 h incubation. All m/z values are quoted as nominal masses.

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FIG. 2. Effect of NH4OH treatment on glycopeptide 2. (A) ESI mass spectrum obtained on incubating 1 nmol for 4 h in NH4OH. (B) ESI mass spectrum obtained under similar conditions after 8 h incubation. (C) FAB CID tandem mass spectrum and fragmentation pattern for [M 1 H]1 ion m/z 829 produced after incubation for 4 h. All m/z values are quoted as nominal masses.

ment is located at the amino acid residue to which the glycan was previously attached. We postulate this mass increment to correspond to a product formed during a two-step reaction, in which the glycan is released from the serine by b-elimination to yield dehydroalanine (Dha) (13), and NH3 is subsequently added across the double bond of the Dha by a Michael-type addition reaction (Scheme 2). It is also clear from the CID spectrum that the C-terminal amide is retained after treatment of the glycopeptide with NH4OH, while treatment with NaOH causes the amide to be cleaved and converted to the free acid. Somewhat more complex behavior is observed for glycopeptides 4 and 5 on b-elimination using NH4OH. Glycopeptide 4 contains a single glycan moiety attached to the serine residue adjacent to the internal proline, while glycopeptide 5 contains three carbohydrate moieties attached to consecutive serine residues. The ESI mass spectra obtained on treatment of glycopeptide 4 with NH4OH for 1, 2, 4, 6, or 8 h (not shown) clearly demonstrate that b-elimination of the glycan and addition of NH3 across the double bond occur in two separate steps. The spectra contain two pseudomolecular ions; that at m/z 834, corresponding to [rPep 1

Na]1, decreases in relative abundance with longer incubation times, while that at m/z 851, corresponding to [rPep 1 NH3 1 Na]1, increases in relative abundance on longer incubation. We suggest that this delay in addition of NH3 to the Dha formed adjacent to the internal proline residue may be for steric reasons (see below). The ESI spectra obtained following incubation of glycopeptide 5 with NH4OH for 1, 2, 4, 6, and 8 h (not shown) make it clear that several different rPep products are formed. The intensities of the [M 1 Na]1 pseudomolecular ions corresponding to the different rPep species detected (m/z 1447 for glycopeptide 5, m/z 1243 for [5 minus 1 GalNAc 1 17], m/z 1039 for [5 minus 2 GalNAc 1 2 3 17], and m/z 818 for [5 minus 3 GalNAc 1 2 3 17]) were studied as a function of reaction time. From these experiments it is readily observed that efficient removal of one glycan moiety occurs almost immediately, with the intensity of m/z 1243 reaching a maximum within approximately 1–2 h. After about 6 h none of the intact glycopeptide 5 is detectable, and the most intense ion represents a species from which two of the glycan moieties have been eliminated. After 8 h the amount of the glycopeptide species retaining only one glycan moiety is further

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FIG. 2—Continued

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SCHEME 2. Proposed mechanism for release of peptide from Olinked glycopeptide using NH4OH.

decreased because of the removal of the third and final glycan. It is noteworthy that although removal of all three glycans occurs, addition of predominantly only two molecules of NH3 is observed after 8 h. We assume that this effect is similar to that described by us previously (13), when although elimination of the glycan from the amino acid residue adjacent to the internal proline proceeds, reduction of the resulting double bond does not. We suggest that this observation may be explained by steric hindrance of the double bond formed, due to its proximity to a proline imide bond. The ESI mass spectra (not shown) from glycopeptides 6, 7, and 8, obtained following b-elimination for 4 h using NH4OH, contain pseudomolecular ions corresponding to [rPep 1 Na]1 (m/z 862) and [rPep 1 17 1 Na]1 (m/z 879). The m/z value at which the [rPep 1 Na]1 ions appear indicates that the threonine residue in the released peptide has been converted into a dehydrobutyric acid (Dhb) residue. The [rPep 1 17 1 Na]1 ion results from the addition of NH3 across the double bond of the Dhb residue. Additional signals with m/z values 44 amu lower than those for all the pseudomolecular ions were observed. The 244-amu ions can be rationalized as arising from the elimination of CH25CHOH from the threonine side chains during the b-elimination reaction. The relative intensities of these signals were found to depend on reaction time, with each threonine residue present being apparently eventually able to undergo the elimination reaction. The results of similar experiments carried out on glycopeptides 9, 10, and 11 (the Gal-GalNAc-bearing an-

alogues of 6, 7, and 8) are indistinguishable from those obtained from their GalNAc-bearing counterparts. To optimize the conditions yielding maximum amounts of rPep, the duration of the b-elimination reaction using NH4OH was varied from 0 to 36 h for all glycopeptides. The relative amount of rPep obtained from the reactions terminated after 0, 4, 8, 12, 16, 20, 24, and 36 h was determined using ESI–MS by summing the extracted ion profiles for the [rPep 1 Na]1, [rPep 1 17 1 H]1, and [rPep 1 17 1 Na]1 ions. The consumption of glycopeptide (GPep) during the b-elimination reaction was also monitored by summing the extracted ion profiles corresponding to [GPep 1 H]1 and [GPep 1 Na]1. An internal standard was added to each of the samples following termination of the b-elimination reaction and prior to injection into the ESI mass spectrometer to compensate for instrument drift and for suppression effects due to impurities introduced into the sample during the b-elimination reaction. It can be seen that b-elimination of glycopeptide 2 (Fig. 3A) reaches an optimum after approximately 8 h. Similar profiles were obtained for glycopeptides 3 and 4. A rather more complex set of profiles was observed for glycopeptides 6, 7 (Fig. 3B), and 8, which were optimally released after more extensive incubation, probably because the threonine-bound O-glycan is less accessible to the reagent because of the methyl group present on the threonine side chain that is absent from that of serine. From the time-course data, it is clear that the structure of the glycopeptide, and particularly the site of glycan attachment, influences the incubation time which results in maximum yields of the released peptide. For example, the peptide from the serine-bound glycopeptide 2 is optimally released after an incubation time of approximately 8 h, while the peptide originating from the analogous threonine-bound glycopeptide 7 is optimally released after 18 h. In addition, structures in which the site of glycan attachment is sterically hindered, such as 8 and 4, seem to require rather longer incubation times than their less hindered analogues. However, in spite of these variations in optimum incubation time, the results of the time-course studies make it clear that a generally applicable protocol for the treatment of glycopeptides of unknown structure can be arrived at, since incubating for longer than the optimum release time results in only a slow decrease in the amount of rPep when using NH4OH as the b-elimination reagent. We thus recommend that a glycopeptide of unknown structure, available in amounts too small to allow optimization, should be treated with 25% (w/v) NH4OH, at 45°C for 15–16 h. Under these conditions, a sufficient yield of released peptide from any structure should be obtained to allow MS–MS determination of the site of modification and thus the former site of glycan attachment.

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FIG. 3. Effect of incubation time on release of O-linked glycans using NH4OH. (A) Results for glycopeptide 2. (B) Results for glycopeptide 7. Each time point was generated from data obtained on incubation of 1 nmol starting material. A constant (approx. 3 nmol) amount of internal standard was added to each sample prior to ESI–MS analysis.

The simplicity of the b-elimination reaction using NH4OH, compared with that using NaOH for determining sites of O-glycosylation, has been demonstrated. It is, however, of interest to make a direct comparison of the minimum detectable amounts of released peptide achieved using the various different b-elimination procedures. Glycopeptide 2 was used as the model glycopeptide for conducting such a comparison. Various amounts of 2 were thus tested in a series of different procedures (Scheme 1), and onethird of the resulting products were subsequently analyzed using ESI–MS. When using NH4OH it is possible to detect released peptide ([rPep 1 17 1 Na]1 at m/z 851) even when starting with as little as 10 pmol of the intact glycopeptide (Fig. 4A), while the spectrum obtained from 100 pmol of 2 is of excellent quality (Fig. 4B). When using NaOH to achieve b-elimination in combination with either a C18 solid-phase extraction column or a Microcon-SCX desalting device, 100 pmol of glycopeptide 2 was not sufficient to detect released peptide ([rPep 1 Na]1 at m/z 834) using ESI–MS (Figs. 4C and 4D). The im-

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proved minimum detectable amount achieved when using NH4OH for the b-elimination reaction can be explained by a number of factors. First, losses of released peptide due to adsorption onto the stationary material of the C18 solid-phase extraction columns or onto the membrane of the Microcon-SCX cartridge are avoided when using the NH4OH procedure, which does not require any subsequent manipulations other than evaporation of the reagent. Such losses of the released peptide during sample purification are especially troublesome when only small amounts of starting material are available. Second, the products resulting from NH4OH treatment seem to be generally cleaner than those resulting from the two NaOH procedures, as is evident from the fact that the reaction products released from 100 pmol of glycopeptide 2 using NH4OH result in a lower total ESI–MS ion current (TIC) than that resulting from the products of the two procedures using NaOH. Finally, it appears that b-elimination using NH4OH is much gentler than that using NaOH as it degrades the released peptide to a much lesser extent. The suitability of b-elimination using NH4OH coupled with mass spectrometric analysis of the released peptides for the determination of the site of O-glycan attachment in base-labile (i.e., serine- or threoninebound) O-glycopeptides of biological interest is clear. Since the treatment is so mild, we assume that it will also be readily applicable to O-glycopeptides in which the glycan is not an aminohexose but, for example, mannose or fucose. However, such samples have, unfortunately, to date, been unavailable to us for testing. From the fact that both the monosaccharide GalNAc and GalNAc-linked disaccharides are released with directly comparable efficiencies, we predict that both longer chain glycans and O-linked GlcNAc residues should also be susceptible to this treatment and therefore that this method will find wider applicability to other base-labile O-linked glycans. To determine the smallest amount of glycopeptide sample necessary to allow peptide release and subsequent successful MS–MS analysis and assignment of the site of O-glycosylation, we set up a series of experiments in which 10, 5, or 1 pmol of glycopeptide 7 was used. It is important to emphasize that this experiment, based on very small amounts of starting material, is necessary if the true sensitivity of our protocol is to be determined. With recent developments in instrument design and ionization technology, mass spectrometric sensitivity is no longer usually the limiting factor in such analyses. The greatest losses and interferences derive from the chemical treatment and sample work-up. It is thus inappropriate to quote sensitivity if an unrealistically large amount of sample is taken and treated chemically and only then is a realistically small aliquot of it analyzed very sensitively; in most

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FIG. 4. Effect of different b-elimination protocols on recovery of released peptide. (A) ESI mass spectrum obtained from 10 pmol compound 2 after incubation in NH4OH (16 h). (B) ESI mass spectrum from 100 pmol 2 after NH4OH incubation (16 h). (C) ESI mass spectrum obtained from 100 pmol 2 after NaOH incubation (4 h) followed by desalting on a C18 solid-phase column. (D) ESI mass spectrum obtained from 100 pmol 2 after NaOH incubation (4 h) followed by desalting using a Microcon-SCX cartridge. After termination of the b-elimination reactions and isolation of the products, these were dissolved in 30 ml of the carrier solution [0.1% formic acid in 50/50 (v/v) water/acetonitrile] and only 10 ml of the resulting solution was introduced into the mass spectrometer. The same dilution and injection routine were followed for all samples. m/z values are quoted as nominal masses.

cases of a biologically derived sample, it is the absolute amount of sample available that is limiting, not instrumental performance. Each of the samples (10, 5, and 1 pmol) of the glycopeptide was incubated with NH4OH for 8 h, and the

products were analyzed on the Micromass Q-Tof tandem mass spectrometer using nanospray ionization. The CID spectrum of the 10-pmol sample (Fig. 5A) is of very high quality and contains intense B1, B2, B3-NH3, B4, B6, B7, Y06, Y05, and Y04 fragment ions, allowing the full sequence

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FIG. 5. Limits of sensitivity achievable using NH4OH for release of peptide and mass spectrometric sequence determination of the O-glycosylation site. (A) ESI CID tandem mass spectrum and fragmentation pattern for the [M 1 H]1 ion of peptide released from 10 pmol compound 7. (B) ESI CID tandem mass spectrum and fragmentation pattern for the [M 1 H]1 ion of peptide released from 1 pmol compound 7. Ions marked with an m/z value but not further assigned arise by double cleavage and thus represent internal fragments of the peptide.

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of the peptide to be determined and readily allowing the site of glycan attachment to be located. The CID spectrum yielded by the 1-pmol aliquot of the glycopeptide after release of the peptide (Fig. 5B) also clearly contains all those fragment ions (B2, A3, B4, B6, B7, Y04, Y05, and Y06) necessary to define the site of the modified amino acid residue without ambiguity. CONCLUSIONS

The identification of O-glycosylation sites, usually an extremely challenging problem, can be greatly simplified using the protocol described in this paper. Release of the O-linked glycan chain is achieved using b-elimination, and the resulting peptide, in which the former site of O-glycan attachment is marked by a modified amino acid residue, may be sequenced using mass spectrometry to allow positive and unambiguous identification of the site of glycosylation. Clearly, the use of NH4OH greatly enhances the sensitivity of the b-elimination procedure, as well as obviating the need for sample clean-up. The fact that the reaction sometimes yields two rather than one reaction product does not hamper analysis, but can be regarded as an aid in correctly identifying the ions carrying information on former sites of O-glycosylation. The significant increase in chemical sensitivity, combined with recent developments in mass spectrometric techniques (in both ionization and mass analysis), means that the identification of the site of O-glycosylation is now fully compatible with sample quantities usually available. It should be stressed that this picomolar sensitivity is achieved by using picomolar amounts of starting material, rather than starting with large quantities and using small fractions of that for analysis. With its low picomolar sensitivity, this protocol can be considered an extremely valuable tool for glycopeptide analysis. As only a small fraction of the 1-pmol sample was loaded into the nanospray capillary, a further increase in sensitivity to the high femtomolar level can be envisaged, based on the data presented here. ACKNOWLEGMENTS We gratefully acknowledge financial support from The Netherlands Organisation for Scientific Research (NWO), the European Commission’s Human Capital and Mobility Programme (to J. E.T.-O. for S.A.P.), and the European Community Science Project Grant SCI-CT92-0765 (to Hans Paulsen for A.K.).

REFERENCES 1. Bause, E., and Lehle, L. (1979) Eur. J. Biochem. 101, 531–540. 2. Lehle, L., and Bause, E. (1984) Biochim. Biophys. Acta 799, 246 –251. 3. Wilson, I. B. H., Gavel, Y., and Von Heyne, G. (1991) Biochem. J. 275, 529 –534. 4. O’Connell, B. C., Hagen, F. K., and Tabak, L. A. (1992) J. Biol. Chem. 267, 25010 –25018.

5. Clausen, H., and Bennet, E. P. (1996) Glycobiology 6, 635– 646. 6. Marth, J. D. (1996) Glycobiology 6, 701–705. 7. Tomita, M., and Marchesi, V. T. (1975) Proc. Natl. Acad. Sci. USA 72, 2964 –2968. 8. Morris, H. R., Panico, M., Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. (1981) Biochem. Biophys. Res. Commun. 101, 623– 631. 9. Biemann, K. (1988) Biomed. Environ. Mass Spectrom. 16, 99 – 111. 10. Medzihradszky, K. F., Gillece-Castro, B. L., Setterini, C. A., Townsend, R. R., Masiarz, F. R., and Burlingame, A. M. (1990) Biomed. Environ. Mass Spectrom. 19, 777–781. 11. Medzihradszky, K. F., Gillece-Castro, B. L., Townsend, R. R., Burlingame, A. L., and Hardy, M. R. (1996) J. Am. Soc. Mass Spectrom. 7, 319 –328. 12. Setterini, C. A., Medzihradsky, K. F., Masiarz, F. R., Burlingame, A. L., Chu, C., and George-Nascimento, C. (1990) Biomed. Environ. Mass Spectrom. 19, 665– 676. 13. Rademaker, G. J., Haverkamp, J., and Thomas-Oates, J. (1993) Org. Mass Spectrom. 28, 1536 –1541. 14. Carlson, D. M. (1968) J. Biol. Chem. 243, 616 – 626. 15. Shimamura, M., Inoue, Y., and Inoue, S. (1984) Arch. Biochem. Biophys. 232, 699 –706. 16. Rademaker, G. J. (1996) Mass Spectrometry: A Modern Approach to Solving Biological Structural Problems, Ph.D. Thesis, Utrecht University. 17. Rademaker, G. J., and Thomas-Oates, J. (1996) in Protein and Peptide Analysis by Mass Spectrometry (Chapman, J. R., Ed.), pp. 231–241, Humana Press, Totowa, NJ. 18. Greis, K. D., Hayes, B. K., Comer, F. I., Kirk, M., Barnes, S., Lowary, T. L., and Hart, G. W. (1996) Anal. Biochem. 234, 38 – 49. 19. Peters, S., Bielfeldt, T., Meldal, M., Bock, K., and Paulsen, H. (1991) Tetrahedron Lett. 32, 5067–5070. 20. Peters, S., Bielfeldt, T., Meldal, M., Bock, K., and Paulsen, H. (1992) J. Chem. Soc. Perkin Trans. I, 1163–1171. 21. Meldal, M., Holm, C. B., Bojesen, G., Jacobsen, M. H., and Holm, A. (1993) Int. J. Pept. Protein Res. 41, 250 –260. 22. Paulsen, H., Bielfeldt, T., Peters, S., Meldal, M., and Bock, K. (1994) Liebigs Ann. Chem., 369 –379. 23. Paulsen, H., Bielfeldt, T., Peters, S., Meldal, M., and Bock, K. (1994) Liebigs Ann. Chem., 381–387. 24. Atherton, E., Clive, D. L. J., and Sheppard, R. C. (1975) J. Am. Chem. Soc. 75, 6584 – 6585. 25. Arshady, R., Atherton, E., Clive, D. J. J., and Sheppard, R. C. (1981) J. Chem. Soc. Perkin Trans. I, 529 –537. 26. Rink, H. (1987) Tetrahedron Lett. 28, 3787–3791. 27. Kisfaludy, L., and Scho¨n, I. (1983) Synthesis, 325–327. 28. Kisfaludy, L., and Scho¨n, I. (1986) Synthesis, 303–305. 29. Atherton, E., Holder, J. L., Meldal, M., Sheppard, R. C., and Valerio, R. M. (1988) J. Chem. Soc. Perkin Trans. I, 2887. 30. Paulsen, H., Peters, S., Bielfeldt, T., Meldal, M., and Bock, K. (1995) Carbohydr. Res. 268, 17–34. 31. Kleen, A., and Paulsen, H. Unpublished results. 32. Granovsky, M., Bielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994) Eur. J. Biochem. 221, 1039 –1046. 33. Brockhausen, I., Toki, D., Brockhausen, J., Peters, S., Bielfeldt, T., Kleen, A., Paulsen, H., Meldal, M., Hagen, F., and Tabak, L. A. (1996) Glycoconj. J. 13, 849 – 856.