Protein Glycosylation Analyzed by Normal-Phase Nano-Liquid

Anal. Chem. 2005, 77, 886-894

Protein Glycosylation Analyzed by Normal-Phase Nano-Liquid Chromatography-Mass Spectrometry of Glycopeptides Manfred Wuhrer,* Carolien A. M. Koeleman, Cornelis H. Hokke, and Andre´ M. Deelder

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands

A new method for the mass spectrometric characterization of site-specific protein glycosylation is presented. Glycoprotein samples were subjected to unspecific proteolysis by Pronase, resulting in glycopeptides with peptide moieties of mostly two to eight amino acids. Resulting (glyco-)peptide samples were resolved by nanoscale normalphase liquid chromatography (LC)-online mass spectrometry (MS). Retention depended on the size of the glycan chain and allowed the separation of identical peptide moieties containing different N-glycan structures. Glycopeptides were analyzed in an ion trap instrument performing repetitive ion isolation/fragmentation cycles. While the MS/MS spectra were dominated by fragmentations of glycosidic linkages, MS3 spectra exhibited cleavages of the peptide backbone and provided information on the peptide sequence and glycan attachment site. When applied to the model glycoproteins ribonuclease B and horseradish peroxidase (HRP), the method provided detailed insights into protein glycosylation and revealed some new features of site-specific glycosylation of HRP. Application of the method to Dolichos biflorus lectin, which has hitherto not been studied with respect to its glycosylation, identified two glycans attached alternatively to its single glycosylation site. Thus, the presented, unique combination of Pronase digestion of glycoproteins, normalphase nano-LC, and multistage MS provides a method for the facile characterization of site-specific protein glycosylation. The most common manner of characterizing protein glycosylation involves the following steps: first, an enzymatic or chemical release of the attached glycans; second, labeling/derivatization of the released glycans in order to facilitate detection, make the glycans suitable for chromatography, or both; third, analysis of the glycans by chromatographic or mass spectrometric techniques.1-3 Using scaled-down solid-phase extraction and advanced * To whom correspondence should be addressed. Tel: +31-71-526-5077. Fax: +31-71-526-6907. E-mail: [email protected]. (1) Rudd, P. M.; Guile, G. R.; Ku ¨ ster, B.; Harvey, D. J.; Opdenakker, G.; Dwek, R. A. Nature 1997, 388, 205-207. (2) Callewaert, N.; Van Vlierberghe, H.; Van Hecke, A.; Laroy, W.; Delanghe, J.; Contreras, R. Nat. Med. 2004, 10, 429-434. (3) Rudd, P. M.; Colominas, C.; Royle, L.; Murphy, N.; Hart, E.; Merry, A. H.; Hebestreit, H. F.; Dwek, R. A. Proteomics 2001, 1, 285-294.

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nanoliquid chromatography-mass spectrometry techniques, these approaches exhibit a low-femtomole detection limit and can be integrated in modern proteomics-type approaches.4-7 Major drawbacks are, however, that no information on the glycan attachment sites is obtained and that glycan release is often selective and incomplete. An alternative approach is the generation of (glyco-)peptides by specific endoproteinases or chemical cleavage methods, (glyco-)peptide fractionation by reversed-phase HPLC separation or affinity chromatography using immobilized antibodies or lectins, followed by the characterization of glycopeptide-containing fractions using mass spectrometry, and Edman degradation, as well as exoglycosidases and enzymes for glycan release.8-11 Major limitations of these methods arise from the cleavage step: trypsin, for example, fails to act on many highly posttranslationally modified proteins.12,13 In addition, glycopeptides obtained by selective cleavage methods are often very heterogeneous in size, and peptide moieties may still contain several glycosylation sites, which complicates mass spectrometric analysis.14,15 These problems can be avoided when analyzing Pronasegenerated glycopeptides. Nonspecific proteolysis of a glycopeptide using Pronase results in rather small peptides of two to eight amino acids. These glycopeptides can be readily detected by mass (4) Kawasaki, N.; Itoh, S.; Ohta, M.; Hayakawa, T. Anal. Biochem. 2003, 316, 15-22. (5) Schulz, B. L.; Packer, N. H.; Karlsson, N. G. Anal.Chem. 2002, 74, 60886097. (6) Wuhrer, M.; Koeleman, C. A. M.; Hokke, C. H.; Deelder, A. M. Int. J. Mass Spectrom. 2004, 232, 51-57. (7) Wuhrer, M.; Koeleman, C. A.; Deelder, A. M.; Hokke, C. H. Anal. Chem. 2004, 76, 833-838. (8) Samyn-Petit, B.; Wajda Dubos, J. P.; Chirat, F.; Coddeville, B.; Demaizieres, G.; Farrer, S.; Slomianny, M. C.; Theisen, M.; Delannoy, P. Eur. J. Biochem. 2003, 270, 3235-3242. (9) Bunkenborg, J.; Pilch, B. J.; Podtelejnikov, A. V.; Wisniewski, J. R. Proteomics 2004, 4, 454-465. (10) Zhang, C.; Doherty-Kirby, A.; Huystee, Rv R.; Lajoie, G. Phytochemistry 2004, 65, 1575-1588. (11) Gray, J. S.; Yang, B. Y.; Montgomery, R. Carbohydr. Res. 1998, 311, 6169. (12) Wang, P.; Li, G.; Granados, R. R. Insect Biochem. Mol. Biol. 2004, 34, 215227. (13) Godl, K.; Johansson, M. E.; Lidell, M. E.; Morgelin, M.; Karlsson, H.; Olson, F. J.; Gum, J. R., Jr.; Kim, Y. S.; Hansson, G. C. J. Biol. Chem. 2002, 277, 47248-47256. (14) Tarelli, E.; Smith, A. C.; Hendry, B. M.; Challacombe, S. J.; Pouria, S. Carbohydr. Res. 2004, 339, 2329-2335. (15) Wuhrer, M.; Hokke, C. H.; Deelder, A. M. Rapid Commun. Mass Spectrom. 2004, 18, 1741-1748. 10.1021/ac048619x CCC: $30.25

© 2005 American Chemical Society Published on Web 12/23/2004

spectrometry (MS), as has been shown in a recent report based on MALDI-Fourier transform-ion cyclotron-MS.16 We here describe a method that allows sensitive normal-phase nano-LC-MSbased detection of pronase-generated glycopeptides. A major advantageous feature of the normal-phase system is that retention of the glycopeptide increases with the size of the glycan, which results in the separation of different glycoforms of the same peptide moiety. In reversed-phase chromatography of glycopeptides, in contrast, effects of the carbohydrate moieties on retention are much less pronounced, and often only partial separation of different glycoforms is achieved. The normal-phase nano-LC-MS(/MS) analysis presented in this work can be performed on low-nanogram amounts of Pronase-treated glycoprotein samples without further workup, and online electrospray-ion trap MS allows the detailed characterization of both glycan and peptide moiety using repetitive ion isolation/fragmentation cycles. EXPERIMENTAL SECTION Glycoprotein Cleavage with Pronase. Ribonuclease B (RNase B; 10 µg) obtained from Sigma (Zwijndrecht, The Netherlands) was incubated with Pronase (mixture of proteases from Streptococcus griseus; type XIV bacterial; P-5147; Sigma) at a enzyme/ substrate ratio of 1:100 in 50 mM ammonium hydrogen carbonate containing 5 mM calcium chloride, at 37 °C overnight. Horseradish peroxidase (HRP; Sigma) was likewise digested at enzyme/ substrate ratios of 1:10 and 1:100. The cleavage of Dolichos biflorus lectin (Sigma) was similarly performed at an enzyme/substrate ratio of 1:20 with 4-h incubation. Nano-Liquid Chromatography-Mass Spectrometry. Glycopeptides were separated on a nanoscale Amide-80 column (5 µm; 80 Å; 75 µm × 100 mm; Tosohaas, Montgomeryville, PA) packed by Grom Analytik (Rottenburg, Germany) using an Ultimate nano-LC system (Dionex/LC Packings, Amsterdam, The Netherlands). Solvent A was 0.5% formic acid. Solvent B was 96.5% acetonitrile, 3% water, and 0.5% formic acid. The following gradient conditions were used: t ) 0 min, 87% solvent B; t ) 22 min, 30% solvent B; t ) 25 min, 30% solvent B; t ) 26 min, 87% solvent B. The flow was ∼300 nL/min. Samples (corresponding to 50 ng of glycoprotein) were injected in 84% acetonitrile, 0.5% formic acid.17 The system was directly coupled to an Esquire high-capacity trap ESI-IT-MS (Bruker Daltonik, Bremen, Germany) equipped with an online nanospray source operating in the positive-ion mode. For electrospray (900-1200 V), capillaries (360-µm o.d., 20-µm i.d. with 10-µm opening) from New Objective (Cambridge, MA) were used. The solvent was evaporated at 100 °C with a nitrogen stream of 6 L/min. Ions from m/z 50 to 2000 were registered. Automatic fragment ion analysis was enabled, resulting in MS/ MS spectra of the most abundant peaks. The major ions of each MS/MS spectrum were subjected to an additional ion isolation/ fragmentation cycle, resulting in MS3 fragment ion spectra. Data Interpretation. LC-MS/MS data were screened for glycopeptides using the neutral loss chromatograms indicative of terminal monosaccharides in different charge states (e. g., neutral losses of 81 and 73 are indicative of a double-charged precursor containing terminal hexose and deoxyhexose, respectively). (16) An, H. J.; Peavy, T. R.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 2003, 75, 5628-5637. (17) Garner, B.; Merry, A. H.; Royle, L.; Harvey, D. J.; Rudd, P. M.; Thillet, J. J. Biol. Chem. 2001, 276, 22200-22208.

Alternatively, MS/MS data were screened for characteristic fragment ions, e.g., of m/z 366 ([HexNAc1Hex1 + H]+). For each detected glycopeptide, the glycan composition was deduced from the MS/MS data. This information was used to determine the masses of the peptide moieties. With this list of peptide masses, the sequences of the corresponding glycoproteins RNase B (SwissProt P00656), HRP (SwissProt P00433), and D. biflorus lectin (SwissProt P05045) were scanned for matching random peptide moieties using the FindPept tool (http://www.expasy.org/tools/ findpept.html). Peptide sequences with N-glycosylation sites, which were indicated by this tool, were then compared with the peptide sequence information obtained from MS3 spectra of the glycopeptide species.

RESULTS The following new approach for the characterization of protein glycosylation was tested: glycoproteins were degraded with Pronase, and the resulting glycopeptides, which exhibited small peptide moieties, were fractionated on a nanoscale amide column coupled online to an ion trap mass spectrometer. Samples were analyzed by repetitive ion isolation/fragmentation cycles. Glycopeptides were indicated by neutral loss monitoring based on the obtained MS/MS data. This approach was applied to the model glycoproteins bovine ribonuclease B (RNase B) and HRP, as well as to D. biflorus lectin, of which the glycosylation has not been described yet. For all three glycoproteins, the approach revealed detailed information on glycan compositions as well as glycan attachment sites. Normal-Phase Nano-LC-MS of RNase B Glycopeptides. An aliquot (50 ng) of a Pronase digest of RNase B was analyzed by LC-MS, and obtained data were screened for glycopeptides by looking for neutral loss (NL) of terminal monosaccharide units. The two NL chromatograms of 81 m/z units (NL of one hexose from a double-charged precursor) and 162 m/z units (NL of two hexoses from a double-charged precursor) revealed the elution of glycopeptides between 30 and 38 min. (Figure 1A). The sum spectrum of this region is shown in Figure 2. Based on the automatically acquired MS/MS spectra, the glycan moiety of each individual glycopeptide could be deduced (see Figure 3). The fragment ion spectra were dominated by ions arising from fragmentation of glycosidic linkages, with the most intense signal normally arising from cleavage of the chitobiose core, leaving only one GlcNAc on the peptide moiety (ion at m/z 492 in Figure 3). With the knowledge of the glycan composition, the deduced mass of the peptide moiety was used to search the RNase B protein sequence (SwissProt P00656) for a match by running the program FindPept. In this way, the double-charged ions of the glycopeptide region (Figure 2.) were assigned to the peptides R59N60, N60LT62, S58RN60, N60LTR63, and N60LTRDR65, carrying N-glycans of the composition HexNAc2Hex5-9. Elution positions of the glycopeptides N60-T62 substituted with various high-mannose glycans (Man5-Man9) are indicated in Figure 1B for glycopeptides with peptide moieties N60LT62. The increasing retention with each added monosaccharide residue leads to a separation based on glycan size, similar to what has been described for the separation of oligosaccharide species on an amide column. In addition, variation in the peptide moieties had a considerable influence on the retention times: While N60LT62 carrying a Man5 glycan eluted Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 1. Normal-phase chromatography of N-glycosylated peptides from an RNase B Pronase digest. RNAse B was digested with Pronase, and a 5-ng aliquot was analyzed by normal-phase nano-LC-electrospray mass spectrometry with automatic MS/MS. A screening of fragment ion spectra for a NL of 81 and 162 of one and two hexoses from a double-charged precursor, respectively) as shown in (A) indicated glycosylated species in the region of 30-38 min. Mass spectrometric data (Figures 2 and 3) indicated these species to be small peptides carrying the oligomannosidic Man5-Man9 N-glycan species known to occur on RNase B. Extracted ion chromatograms representing the double-protonated species of glycopeptides with an N60LT62 peptide moiety are given in (B). BPC, base peak chromatogram. A mass spectrum of the glycopeptide region as indicated by a horizontal bar is given in Figure 2.

Figure 2. Mass spectrum of pronase-generated glycopeptides of RNase B Sum spectrum over the whole range of detected glycopeptides obtained from RNase B by Pronase digestion, as indicated in Figure 1. The ions assigned in Figure 2 correspond to double-protonated glycopeptide species. The identified peptide moieties are located in the region S58RN60TLRDR65 of the RNase B sequence (SwissProt P00656), all of them spanning glycosylation site N60. Man5, peptide modification by a pentamannosidic N-glycan structure, etc.

at 31 min (Figure 1B), the other Man5-substituted peptides eluted several minutes later at 33 (N60LTR63), 35 (R59N60), and 35.5 min (S58RN60 and N60LTRDR65). Taken together, the observed profile of glycopeptides fits well with the pattern of high-mannose N-glycans attached to N60 as it is described for RNase B.16,18 Normal-Phase Nano-LC-MS of HRP Glycopeptides. The mass spectrometric data set obtained for a Pronase digest of HRP 888 Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

(enzyme/substrate ratio of 1:10) was screened for fragment ions indicative of various terminal monosaccharides by NL chromatograms. In agreement with the major oligosaccharide species of HRP, which are xylosylated, R1-3-fucosylated trimannosyl N-linked glycans,7,11,19,20 this screening revealed mainly precursors that lost (18) Fu, D.; Chen, L.; O’Neill, R. A. Carbohydr. Res. 1994, 261, 173-186.

Figure 3. Fragment ion spectra of the Pronase-generated glycopeptide R59-N60 of RNase B with a Man5 N-glycan structure. Fragment ion spectrum (MS2) of the double-protonated precursor at m/z 753 (Figure 2). Heterogeneity in mannose content is indicated by double-headed arrows. Pep, peptide moiety.

Figure 4. Normal-phase chromatography of N-glycosylated peptides from an HRP Pronase digest. HRP was digested with Pronase, and a 50-ng aliquot was analyzed by normal-phase nano-LC-electrospray mass spectrometry with automatic MS/MS. Fragment ion spectra were screened for neutral loss of fucose (NL m/z 146), fucosylated GlcNAc (m/z 349), fucosylated GlcNAc with ammonia arising from Asn side-chain cleavage (m/z 366), mannose (m/z 162), and xylose (m/z 132), as well as two fragments arising from chitobiose cleavage with (m/z 967) or without (m/z 821) concomitant loss of core fucose. Mass spectra of the glycopeptide regions as indicated by the horizontal bars are given in Figures 5 and 7. The extracted ion chromatograms (EIC) shown in the inset represent Gly-Asn dipeptides substituted with different glycan chains (see text and Figure 5A). BPC, base peak chromatogram.

the terminal sugars fucose (NL 146), mannose (NL 162), and xylose (NL 132) in MS/MS analyses (Figure 4). Furthermore, neutral loss of a Man3GlcNAc1Xyl1 unit (NL 821), resulting from cleavage between the two core GlcNAc residues, was monitored. This cleavage could occur together with a loss of core fucose (NL 967; Figure 4). Taken together, these cleavages indicated the detection of single-charged HRP glycopeptides in the range from 27 to 32 min (Figure 4). The sum mass spectrum of this time range (Figure 5A) was assigned based on MS/MS spectra of (19) Yang, B. Y.; Gray, J. S.; Montgomery, R. Carbohydr. Res. 1996, 287, 203212. (20) Takahashi, N.; Lee, K. B.; Nakagawa, H.; Tsukamoto, Y.; Masuda, K.; Lee, Y. C. Anal. Biochem. 1998, 255, 183-187.

glycopeptides, which revealed the composition of the glycan moiety and thus allowed the deduction of the mass of the peptide part. Furthermore, acquired MS3 data often provided information on the sequence of the peptide moiety (Figure 6). These data allowed the assignment of glycans to eight of the nine potential N-glycosylation sites of HRP (Table 1). To get information on the glycosylation of site N216, a less complete Pronase digest of HRP (enzyme/substrate ratio of 1:100) was analyzed by normal-phase nano-LC-MS (Figure 5B). Glycopeptide species, which now contained slightly longer peptide chains of up to 11 amino acids (triple-charged glycopeptide species G211-Y231 at m/z 788), were detected mainly in double-protonated and triple-protonated form. Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 5. Mass spectra of HRP glycopeptides with large glycans. Mass spectra of the detected glycopeptides with large glycans, which were obtained from HRP by Pronase digestion at a Pronase/HRP ratio of 1:10 (A; protonated species were registered) and 1:100 (B; glycopeptide species with two or three protons were registered). All glycopeptides carry a xylosylated trimannosyl N-glycan, which is fucosylated unless indicated otherwise (-Fuc; no fucose attached). The ion at m/z 1303 corresponds to a glycan attached to a single asparagine (N#), thus not allowing its assignment to a specific N-glycosylation site of HRP. C121-C41-N43; N-glycosylated peptide with a disulfide bridge, as indicated in Figure 9.

Figure 6. Fragment ion spectra of the Pronase-generated glycopeptide S283-N285 of HRP with a fucosylated, xylosylated trimannosyl N-glycan. (A) Fragment ion spectrum (MS2) of the protonated precursor at m/z 1487 (see Figure 5A). The fragment ion at m/z 520 was isolated and fragmented (B), resulting in cleavage of the glycosidic linkage and amide (peptide) bonds, the latter being assigned according to Biemann.30 Furthermore, a ring cleavage (0,2X) of the GlcNAc was observed in the MS3 spectrum (B), leaving only the ring positions C1 and C2 on the peptide moiety.31

Besides several glycopeptide species, which confirmed the glycosylation pattern described above, peptide N216-F217 carrying the Man3GlcNAc2Fuc1Xyl1 N-glycan structure was detected (Figure 5B and Table 1). Most of the ions corresponding to a nonfucosylated glycopeptide, which were observed in Figure 5A, could be unambiguously attributed to in-source decay, as their extracted ion chromatograms coincided with those of the corresponding fucosylated 890

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species. Only the two nonfucosylated glycopeptides assigned to the signals at m/z 1214 and 1327 (Figure 5A) could be identified as real species present in the Pronase digest, indicating the occurrence of a Man3GlcNAc2Xyl1 N-glycan structure at N316. The extracted ion chromatogram of m/z 1214 (inset in Figure 4), which corresponds to the peptide glycine-asparagine carrying a Man3GlcNAc2Xyl1 N-glycan structure, revealed two peaks: an earlier eluting species (retention time 28.6 min), which indicates

Table 1. Obtained Data on the Occupation of HRP N-Glycosylation Sitesa N-glycosylation site

peptide moieties

large glycan moieties (mass, m/z)

small glycan moieties (mass, m/z)

Asn43 Asn87 Asn188 Asn216 Asn228 Asn244 Asn285

C121sC41PN43 D86NT88 N188R189 N216-F217* N228-T229 G243-N244 S283PNA286 S283PN285 N285AT287 N298-ST300 N298-S299 G315-NI317 G315-N316

Man3GlcNAc2Fuc1Xyl1 (1622) Man3GlcNAc2Fuc1Xyl1 (1519) Man3GlcNAc2Fuc1Xyl1 (1459) Man3GlcNAc2Fuc1Xyl1 (725)* Man3GlcNAc2Fuc1Xyl1 (1404) Man3GlcNAc2Fuc1Xyl1 (1360) Man3GlcNAc2Fuc1Xyl1 (1558) Man3GlcNAc2Fuc1Xyl1 (1487) Man3GlcNAc2Fuc1Xyl1 (1475)

GlcNAc1Fuc1 (801)

Asn298 Asn316

Man3GlcNAc2Fuc1Xyl1 (1390) Man3GlcNAc2Xyl1 (1327) Man3GlcNAc2Xyl1 (1214)

GlcNAc1Fuc1 (638)

GlcNAc1Fuc1 (737) GlcNAc1Fuc1 (666) GlcNAc1Fuc1 (670) GlcNAc1Fuc1 (569)

a Data are taken from the LC-MS run of the sample with Pronase/HRP ratio of 1:10 (Figure 5A), except for site Asn 216 (/), for which data were obtained after Pronase digestion with a Pronase/HRP ratio of 1:100 (Figure 5B).

Figure 7. Mass spectrum of Pronase-generated glycopeptides of HRP carrying a disaccharide. Sum spectrum over the range of Fuc1GlcNAc1substituted glycopeptides obtained from HRP by Pronase digestion, as indicated in Figure 4. Besides proton adducts, potassium adducts were registered, which are marked by asterisks. The ion at m/z 520 corresponds to a disaccharide attached to a single asparagine (N#), thus not allowing its assignment to a specific N-glycosylation site of HRP.

the occurrence of this glycopeptide in the Pronase digest (peptide G315-N316 carrying a Man3GlcNAc2Xyl1 N-glycan, as well as a second signal of the same mass (retention time 29.3 min), which seems to arise from in-source defucosylation of the peptide G243N244 carrying the Man3GlcNAc2Fuc1Xyl1 glycan (m/z 1360; inset Figure 4 and Figure 5A). For the ion at m/z 1327 (Man3GlcNAc2Xyl1 on G315-I317), no fucosylated counterpart was detected (Figure 5A). Besides glycopeptides with N-glycan structures of five and more monosaccharides, earlier-eluting glycopeptides with smaller glycan moieties were indicated by the neutral loss 146 chromatogram (Figure 4). A mass spectrum of the corresponding elution range revealed a variety of mainly protonated glycopeptides (Figure 7). MS/MS data indicated that these glycopeptides contained disaccharide units consisting of one HexNAc and one deoxyhexose (fucose; Figures 8A and 9A). Again, analysis of the glycopeptides by repetitive ion isolation/fragmentation cycles resulted in MS3 spectra that provided particularly valuable information on the peptide moiety (Figures 8B,C and 9B, C). In addition, MS/MS spectra of these early-eluting glycopeptides consistently indicated loss of HexNAc1deoxyhexose1 disaccharides

by cleavage of either the N-glycosidic bond or the Asn side-chain amide bond, resulting in neutral losses of m/z 349 and 366, respectively, as indicated in Figure 4. These data allowed the assignment of the disaccharide units to four of the nine potential N-glycosylation sites of HRP (Table 1). Our recent study of HRP glycosylation by MALDI-TOF/TOF-MS of tryptic glycopeptides has likewise indicated HexNAc1deoxyhexose1 disaccharide units on HRP, but only indicated this modification at two N-glycosylation sites (namely N285 and N298).15 Normal-Phase Nano-LC-MS of Glycopeptides from D. biflorus Lectin. LC-MS data obtained with a Pronase digest of D. biflorus lectin were screened for glycopeptides by searching MS/MS spectra for fragments of m/z 366 ([HexNAc1Hex1 + H]+) or 528 ([HexNAc1Hex2 + H]+) (Figure 10A). Two glycopeptides were detected both in double-charged (not shown) and singlecharged forms (Figure 10B and C). The double-protonated species of these glycopeptides were further analyzed by automatic MS/ MS, which indicated glycan compositions of Hex3HexNAc2dHex1Pent1 (Figure 11A, double protonated species of m/z 753, corresponding to single-protonated species of m/z 1504 in Figure 10C) and Hex5HexNAc2 (Figure 11B, double-protonated species Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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Figure 8. Fragment ion spectra of the Pronase-generated glycopeptide S298-N300 of HRP with a GlcNAc1Fuc1 N-glycan. (A) Fragment ion spectrum (MS2) of the protonated precursor at m/z 670 (see Figure 7). The major two fragment ions were isolated and fragmented (B, C), giving rise to fragmentation of glycosidic linkages and amide bonds.

Figure 9. Fragment ion spectra of the Pronase-generated glycopeptide C41-N43 disulfide-linked to C121 of HRP carrying a GlcNAc1Fuc1 N-glycan. (A) Fragment ion spectrum (MS2) of the protonated precursor at m/z 801 (see Figure 7). The major two fragment ions were isolated and fragmented (B, C), giving rise to fragmentation of glycosidic linkages and amide (peptide) bonds. Losses arising from fragmentation of the cystine are indicated by double-headed arrows.

of m/z 775, corresponding to single-protonated species of m/z 1550 in Figure 10C). MS/MS spectra were dominated by fragment ions at m/z 537, which corresponded to the single-protonated peptide moiety retaining one HexNAc residue. For both glycopeptides, the registered mass of the protonated peptide moiety after loss of the glycan part was 334.1 Da (Figure 11). Using the program FindPept, the peptide moiety was identified as the tripeptide N136NS138 of D. biflorus lectin (SwissProt P05045), which comprised the potential N-glycosylation site N136 of this protein. Taken together, two different N-glycan structures may occur at 892 Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

N136 of D. biflorus lectin, i.e., Hex3HexNAc2dHex1Pent1, which is most likely a xylosylated, core-fucosylated trimannosyl N-glycan commonly found on plant glycoproteins or, alternatively Hex5HexNAc2, which may be interpreted as an oligomannosidic structure. DISCUSSION The presented method of Pronase digestion of a glycoprotein sample followed by normal-phase nano-LC multistage MS/MS on an ion trap instrument is suitable to obtain information on both

Figure 10. Normal-phase LC-MS/MS of N-glycosylated peptides from an Pronase digest of D. biflorus lectin. D. biflorus lectin was digested with Pronase, and a 250-ng aliquot was analyzed by normal-phase nano-LC-electrospray mass spectrometry with automatic MS/MS. Fragment ion spectra were screened by extracted ion chromatograms for characteristic B-ions of m/z 366 ([HexNAc1Hex1 + H]+) and 528 [HexNAc1Hex1 + H]+ (A). For the two detected glycopeptides, EIC of the MS data (B) as well as a sum mass spectrum of the relevant elution range (indicated by a horizontal bar) (C) are given. #, proton adduct; 4, sodium adduct; O, potassium adduct.

Figure 11. Fragment ion spectra of the Pronase-generated glycopeptides N136NS138 of D. biflorus lectin with two different glycan moieties. Fragment ion spectra (MS2) of the double-protonated precursors at m/z 752 and 775, which correspond to the single-protonated species (Figure 10C) at m/z 1504 and 1550, respectively. From the MS2 spectra, the glycan moieties of the glycopeptides were deduced as Hex3HexNAc2dHex1Pent1 and Hex5HexNAc2, respectively. The deduced mass of the peptide moiety (334.1 Da) allowed its identification as peptide N136NS138 of the D. biflorus lectin. dHex, deoxyhexose; Pent, Pentose.

glycan attachment sites and glycan composition. When compared to conventional methods for the analysis of site-specific glycosylation, the presented approach exhibits several advantages: 1. Pronase Treatment. this unspecific proteinase mixture generates glycopeptides with small peptide moieties, which can

be readily analyzed by mass spectrometry. Other cleavage methods, which exhibit a higher specificity, for example, treatment with trypsin, often lead to large peptide moieties that can contain several glycosylation sites. This can complicate the assignment of glycans to individual glycosylation sites. Tryptic cleavage of Analytical Chemistry, Vol. 77, No. 3, February 1, 2005

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HRP, for example, results in a cleavage product containing two N-glycosylation sites.11,15 It should be noted that the broad specificity of the Pronase proteinase mixture results in a heterogeneity of cleavage products. For example, five different peptide moieties were detected in the Pronase digest of RNase B (Figure 2), and analysis of a Pronase digest of chicken ovalbumin by Fourier transform-ion cyclotron-MS resulted in the detection of glycosylated versions of six different peptide moieties spanning one single glycosylation site.16 Hence, interpretation of the MS data obtained in the case of a glycoprotein with an unknown number of glycosylation sites and not yet defined carbohydrate substituents could eventually be rather tedious. Furthermore, when applied to a glycoprotein of unknown amino acid sequence, the here presented pronase-based approach should be accompanied by other methods such as MS analysis of tryptic peptides in order to allow protein identification. 2. Normal-Phase Chromatography. Normal-phase chromatography using an amide column has been successfully applied to the separation of glycans1,3,6,7,21 as well as peptides.22-24 In both cases, interactions seem to be mainly based on hydrogen bonds. When analyzing Pronase-generated glycopeptides, retention is mainly due to the glycan moiety, with glycopeptides of similar glycan composition eluting in a narrow time window (compare Figures 1 and 4). This allows the identification of glycopeptide species based on their late elution as compared to other Pronasegenerated cleavage products. The specific retention of glycopeptides on normal-phase material has recently been used for solid-phase extraction of glycopeptides from glycoprotein digests.25 Furthermore, normal-phase HPLCsin contrast to reversed-phase chromatography, which is conventionally performed with glycopeptidessresults in the efficient size separation of different glycosylation variants of a particular peptide moiety (Figures 1 and 4). The set of chromatographic data was successfully used for the interpretation of the mass spectrometric data, allowing a discrimination of real glycopeptide species from those arising from in-source decay. This possibility is a clear advantage (21) Tolstikov, V. V.; Fiehn, O. Anal. Biochem. 2002, 301, 298-307. (22) Yoshida, T. J. Biochem. Biophys. Methods 2004, 60, 265-280. (23) Yoshida, T.; Okada, T. J. Chromatogr., A 1999, 841, 19-32. (24) Yoshida, T. Anal. Chem. 1997, 69, 3038-3043. (25) Hagglund, P.; Bunkenborg, J.; Elortza, F.; Jensen, O. N.; Roepstorff, P. J. Proteome Res. 2004, 3, 556-566. (26) Demelbauer, U. M.; Zehl, M.; Plematl, A.; Allmaier, G.; Rizzi, A. Rapid Commun. Mass Spectrom. 2004, 18, 1575-1582. (27) Jebanathirajah, J.; Steen, H.; Roepstorff, P. J. Am. Soc. Mass Spectrom. 2003, 14, 777-784. (28) Wang, F.; Nakouzi, A.; Angeletti, R. H.; Casadevall, A. Anal. Biochem. 2003, 314, 266-280. (29) Henriksson, H.; Denman, S. E.; Campuzano, I. D.; Ademark, P.; Master, E. R.; Teeri, T. T.; Brumer, H., III. Biochem. J. 2003, 375, 61-73. (30) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111. (31) Domon, B.; Costello, C. Glycoconjugate J. 1988, 5, 253-257.

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of the normal-phase separation system when compared to reversedphase systems. 3. Multistage MS/MS. Scanning of the MS/MS data for glycopeptides using neutral-loss chromatograms and extracted ion chromatograms of oxonium ions arrising from glycan chain fragmentation readily highlights glycopeptide species. Interpretation of the MS/MS spectra allows the deduction of the glycan composition as well as the mass of the peptide moiety. By trimming the glycan moiety in an MS/MS step down to a single GlcNAc, further MS3 analysis is not dominated any more by the cleavage of glycosidic linkages but provides a complementary data set revealing information on the peptide sequence and the glycan attachment site.26 Thus, our approach overcomes the limitations of conventional LC-MS/MS analyses of glycopeptides using collision-induced fragmentation, where fragment ions are often dominated by cleavages of glycosidic linkages and hardly provide information on the peptide moieties.27-29 The efficacy of the presented method is apparent from the detailed picture of glycosylation obtained for RNase B and HRP. Particularly for HRP, the chosen approach not only confirmed the known pattern of glycosylation but provided additional information when compared to literature data:11,19,20 Unusual HexNAc1deoxyhexose1 disaccharides could be assigned to four of the nine potential N-glycosylation sites of HRP (Table 1). Our recent study of HRP glycosylation by MALDI-TOF/TOF-MS has likewise indicated this disaccharide modification to occur on HRP, but detection succeeded only for two of the nine N-glycosylation sites.15 Moreover, information on glycan attachment site and glycan composition was obtained for a sample of hitherto unknown glycosylation. Thus, glycopeptide analysis by normal-phase nano-LC-MS with multistage MS/MS is an efficient tool for the analysis of protein glycosylation. Due to its sensitivity, this method seems to be suitable for the microscale analysis of glycoproteins. Together with other glycoanalytical approaches such as the analysis of enzymatically released glycans,3,5,7 or the analysis of glycopeptides by MALDI-TOF/TOF-MS,15 the presented method should help to further integrate the characterization of protein glycosylation into proteomics-type analytical approaches. Detailed analysis of complex glycoprotein samples, however, should preferably be performed using a combination of different methods including conventional glycan release and the here presented nano-LCMS/MS analysis of Pronase-generated glycopeptides, to allow the integration of complementary data sets.

Received for review September 17, 2004. Accepted November 8, 2004. AC048619X