Man GlcNAc Research Articles

Extraction and purification of a High Mannose type oligosaccharide from Phaseolus lunatus beans by oxidative release with sodium hypochlorite

High Mannose glycans (HM) are intermediates for the eukaryotic glycosylation pathway but are not commonly found on mature glycoproteins. HM, particularly Man9GlcNAc2 (M9), are present on the glycan shield of viral envelopes and have received attention as elements for semi-synthetic vaccines. The most common strategy to produce HM is extensive pronase digestion from soybean agglutinin (SBA) to obtain Asn-M9. Storage proteins from Phaseolus lunatus (butter beans) are abundant in M9 and could also be used for M9 production. Chemical alternatives to enzyme-based protocols such as sodium hypochlorite (NaClO) have been used effectively to release N-glycans. Here we present an application of the NaClO glycan release strategy to produce M9 from Phaseolus lunatus beans as an alternative to SBA and the characterisation of some of the release side products that support the release reaction mechanism.

Analysis of N-glycan profile of Arabidopsis alg3 cell culture.

N-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man3GlcNAc2 (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N-glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture.

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Approaches towards the core pentasaccharide in N-linked glycans

All eukaryotic cells share a common core pentasaccharide, Man3GlcNAc2 (M3), which is found in both dolichol-linked oligosaccharides (DLOs) and N-linked glycoproteins. A pentasaccharide M3 can be elongated and elaborated by various glycosyltransferases and is thus considered as the key intermediate in the in vitro synthetic pathway of N-glycans. In this review, we describe the progresses in the preparation of M3 by chemical and chemoenzymatic methods. We first draw a brief schematic view of the typical N-glycan biological pathway and then focus on the requirements and challenges in obtaining core pentasaccharides. Representative methods and recent reported findings, especially research progress in chemoenzymatic synthesis, are highlighted. In addition, the opportunities in exploiting novel methods for constructing N-glycans, as well as their applications, are discussed.

Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation

UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations and glucosylates from only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically 15N-labeled interleukin 8 (IL-8) C-terminal (34-72) glycopeptides bearing a Man9GlcNAc2 (M9) oligosaccharide. Chemical shift perturbation mapping NMR experiments suggested that Phe65 of the glycopeptide specifically interacts with UGGT. To analyze this interaction, we constructed a glycopeptide library by varying Phe65 with 10 other natural amino acids, via parallel native chemical ligation between a glycopeptide-α-thioester and a peptide library consisting of 11 peptides. UGGT assay against the glycopeptide library revealed that, although less hydrophobic glycopeptides could be used as substrates for UGGT, hydrophobic glycopeptides are preferred.

The neutral N-linked glycans of the ustilaginomycete yeast Sympodiomycopsis paphiopedili.

Sympodiomycopsis paphiopedili is a basidiomycetous yeast under the subphylum Ustilaginomycotina and is a commensal organism originally isolated from the nectar of a plant species in Japan. In this study, the neutral N-linked glycans of S. paphiopedili were prepared and structurally analysed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Glycosidase digestion analyses were also performed to verify certain glycan linkages. HPLC and MS analyses revealed the presence of neutral N-linked glycans ranging from Man3 GlcNAc2 -PA to Man9 GlcNAc2 -PA in length. The most abundant neutral N-linked glycan structure in this species was found to be the Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M8A). Moreover, the second and third most abundant neutral N-linked glycan in S. paphiopedili were the Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M9A) and the Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M3B). On the other hand, the effect of the combination of glycoprotein extraction methods (citrate buffer extraction or bead extraction) and the subsequent glycan release methods (hydrazinolysis or PNGase F digestion) on the detection of N-linked glycan peaks was also examined for S. paphiopedili and Saccharomyces cerevisiae in order to avoid under-representation of N-linked glycan structures. High mannose and possible hypermannosylated glycan peaks were detected in all method combinations in S. cerevisiae with the citrate buffer extraction-hydrazinolysis method giving the highest peak yields as compared with the other methods. Here we report the first account of the structural analysis of the neutral N-linked glycan of S. paphiopedili and the comparison of the effect of combinations of glycoprotein extraction methods and glycan release methods with that of the glycan analysis in S. paphiopedili and S. cerevisiae. Copyright © 2017 John Wiley & Sons, Ltd.

Endo‐α‐Mannosidase‐Catalyzed Transglycosylation

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1- 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc1 Man9 GlcNAc2 ).

Insights from NMR Spectroscopy into the Conformational Properties of Man-9 and Its Recognition by Two HIV Binding Proteins.

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.

Endoplasmic Reticulum (ER)-Targeted, Galectin-Mediated Retrograde Transport by Using a HaloTag Carrier Protein.

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein. This comprised N-terminal galectin-1 for cell-surface binding (G1), a protease cleavable sequence (ps), a HaloTag domain for attaching exogenous compounds (Halo), and a C-terminal KDEL sequence for ER retention. Fluorescently labeled G1-ps-Halo-KDEL passed through the Golgi apparatus and reached the ER. By using Man9 GlcNAc2 -BODIPY as a cargo compound, the carrier protein was also delivered into the ER with concomitant processing of mannose to Man5,6, by the ER-resident α1,2-mannosidase. G1-ps-Halo-KDEL might serve as a new type of delivery carrier protein to direct compounds into the ER.

Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics.

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality-control systems composed by various proteins. Among them, UDP-glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo-misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene-cysteine moiety on the protein surface (pDHFR) and Man9 GlcNAc2 -methotrexate (M9-MTX). All five M9-MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.

Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand

Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (–OH, –Gly–NH2, and –Gly–Glu–tBu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order –Gly–Glu–tBu > –Gly–NH2 > –OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by the folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system.

Glycopeptide mimetics recapitulate high-mannose-type oligosaccharide binding and function.

High-mannose-type glycans (HMTGs) decorating viral spike proteins are targets for virus neutralization. For carbohydrate-binding proteins, multivalency is important for high avidity binding and potent inhibition. To define the chemical determinants controlling multivalent interactions we designed glycopeptide HMTG mimetics with systematically varied mannose valency and spacing. Using the potent antiviral lectin griffithsin (GRFT) as a model, we identified by NMR spectroscopy, SPR, analytical ultracentrifugation, and microcalorimetry glycopeptides that fully recapitulate the specificity and kinetics of binding to Man9 GlcNAc2 Asn and a synthetic nonamannoside. We find that mannose spacing and valency dictate whether glycopeptides engage GRFT in a face-to-face or an intermolecular binding mode. Surprisingly, although face-to-face interactions are of higher affinity, intermolecular interactions are longer lived. These findings yield key insights into mechanisms involved in glycan-mediated viral inhibition.

Structural and Kinetic Dissection of the endo‐α‐1,2‐Mannanase Activity of Bacterial GH99 Glycoside Hydrolases from Bacteroides spp.

Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1-3 Man9 GlcNAc2 ), releasing mono-, di- and triglucosylated-mannose (Glc1-3 -1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan→1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol(-1) preference for mannose-configured substrates in the -2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates.

Endoplasmic reticulum lectin XTP3‐B inhibits endoplasmic reticulum‐associated degradation of a misfolded α1‐antitrypsin variant

The endoplasmic reticulum (ER) is an organelle that synthesizes many secretory and membrane proteins. However, proteins often fold incorrectly. Terminally misfolded polypeptides in the ER are retro-translocated to the cytosol, where they are ultimately degraded by the ubiquitin-proteasome system, a process termed ER-associated degradation (ERAD). By recognizing the specific structures of N-linked oligosaccharides attached to polypeptides, lectins play an important role in the quality control of glycoproteins in the ER. Mammalian OS-9 and XTP3-B are ER-resident lectins that contain mannose 6-phosphate receptor homology (MRH) domains, which recognize sugar moieties; OS-9 has one MRH domain and XTP3-B has two. Both are involved in ERAD, but the functional differences between the two are poorly understood. The present study analyzed the function of human XTP3-B, and found, by frontal affinity chromatography analysis, that its C-terminal MRH domain specifically recognized the Man9 GlcNAc2 (M9) glycan invitro and M9 glycans on an ERAD substrate NHK, a terminally misfolded α1-antitrypsin variant, invivo. Furthermore, endogenous XTP3-B was a component of the HRD1-SEL1L membrane-embedded ubiquitin ligase complex, an association that was stabilized by a direct interaction with SEL1L. The lectin activity of XTP3-B was required for its binding to NHK, but not for its association with SEL1L. Unlike OS-9, XTP3-B did not enhance the degradation of misfolded glycoproteins, but instead inhibited the degradation of NHK bearing M9 oligosaccharides. Therefore, we propose that XTP3-B recognizes M9 glycans on unfolded polypeptides, thereby acting as a negative regulator of ERAD, and also protects newly synthesized immature polypeptides from premature degradation.

Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides.

High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes.

Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe

The early stages of N-linked glycosylation are highly conserved between fungal and mammalian cells. Such N-linked oligosaccharides are synthesized through the ordered assembly of a dolichyl pyrophosphate (Dol-PP)-linked Glc 3Man 9GlcNAc 2 structure by the sequential actions of several glycosyltransferases located in the endoplasmic reticulum (ER). Of the glycosyltransferase genes, Saccharomyces cerevisiae ALG3 has been identified to encode the Dol-P-Man:Man 5GlcNAc 2-PP-Dol α1,3-mannosyltransferase, and an alg3 mutant has been shown to accumulate an Endo H-resistant M5B (Manα1,2-Manα1,2-Manα1,3(Manα1,6-)-Manβ1,4-GlcNAcβ1,4-GlcNAc) structure. Although Schizosaccharomyces pombe contains a homolog of the ALG3 gene (SPAC7D4.06c), the role of this gene in oligosaccharide biosynthesis is not at all clear. In this study, we deleted the alg3 + gene in the och1Δ mutant and analyzed the detailed oligosaccharide structures in alg3Δ och1Δ double mutant. The oligosaccharides were prepared from cell-surface glycoproteins by hydrazinolysis and fluorescent labeling with 2-aminopyridine. The labeled oligosaccharides were analyzed by high performance liquid chromatography, in combination with sequential glycosidase digestion and methylation analysis. These analyses revealed that the N-linked oligosaccharides of S. pombe alg3Δ och1Δ cells mainly consisted of two or three α-galactose-capped M5B structures. Finally, western blot analysis of recombinant human transferrin suggested that heterologously expressed glycoproteins in alg3Δ och1Δ cells have Endo H-resistant N-linked oligosaccharide structures similar to those of alg3Δ och1Δ cell-surface glycoproteins.

Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach

Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5–2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-β-galactosidase. Our results verified the presence of high-mannose-type Man 5GlcNAc 2 compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.

Homogeneous sugar modification improves crystallization of measles virus hemagglutinin

Measles virus (MV) enters cells by binding to the signaling lymphocyte activation molecule (also called CD150) on the cell surface, and thus shows the lymphotropism and immunosuppressive effects. The head domain (residues Asp 149 to Arg 617) of the MV hemagglutinin (MV-H), the attachment protein, was produced using a transient expression system in HEK293T cells. The purified MV-H protein was heterogeneous because of a variety of complex-sugar modifications. The complex-sugar-type MV-H was crystallized successfully, and the crystals belonged to the space group P41212 with the unit cell dimension of a = b = 134 Å, c = 100 Å, but diffracted only to 3.0 Å resolution. MV-H was also expressed in HEK293SGnTI(−) cells lacking the N-acetylglucosaminyltransferase I activity, which render N-linked glycans of the proteins restricted and homogeneous, producing the oligomannose, Man 5GlcNAc 2. The native and selenomethionyl derivative proteins of the oligomannose-type MV-H were crystallized, and the native crystals well diffracted to 2.6 Å resolution. Thus, homogeneous sugar modification may be useful for improved crystallization of heavily sugar-modified viral envelope proteins.

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito.

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.

Characterization of oligosaccharide moieties of glycopeptides by microwave‐assisted partial acid hydrolysis and mass spectrometry

Microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to study oligosaccharide structures of glycopeptides. Tryptic N-glycosylated peptides of horseradish peroxidase, with MH+ ions at m/z 2533, 2612, 3355, 3673, and 5647, were used as test cases. Within a microwave exposure with trifluoroacetic acid of 2 min, partial cleavages of the oligosaccharides of these tryptic N-glycosylated peptides were observed. The data showed that the most labile group within the oligosaccharides is the fucose (Fuc) residue, and that a majority of the end cleavage products are peptides with one N-acetylglucosamine (GlcNAc) residue linked to asparagine (Asn). In addition, the glycopeptides with m/z 3355 and 3673 carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, the glycopeptide at m/z 5647 carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and the glycopeptides at m/z 2612 and 2533 carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the m/z 2612 peptide at Asn286 is partially occupied. This simple and rapid method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.