Usefulness of glycopeptide mapping by liquid chromatography/mass spectrometry in comparability assessment of glycoprotein products.

We previously reported on glycopeptide mapping of erythropoietin (EPO) by liquid chromatography/mass spectrometry (LC/MS). Using this method, glycopeptides in proteolytic digestion can be eluted before peptides, and are further separated on the basis of the carbohydrate structure. The detailed glycosylation at each glycosylation site can be elucidated based on mass chromatography and mass spectroscopy. In this study, we evaluated glycopeptide mapping with regard to its use in comparability assessment of glycoprotein products possessing multiple glycosylation sites. Models of closely related glycoprotein products used in this study are EPOs produced from three different sources. We previously reported that there are differences in the carbohydrate heterogeneity of these EPOs with regard to sialylation, acetylation, and sulphation patterns, using sugar mapping by LC/MS. In this paper, we demonstrated that glycopeptide mapping can distinguish site-specific glycosylation among these three EPOs and reveal the differences in acetylation, sialylation, and sulphation at each glycosylation site in one analysis. Our method can thus be useful in comparability assessment of therapeutic glycoproteins in terms of glycosylation.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

LC/MS analysis of complex multiglycosylated human α1-acid glycoprotein as a model for developing identification and quantitation methods for intact glycopeptide analysis

The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of α₁-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.     

Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.     

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.     

Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms

Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.     

Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.     

Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: analysis of glycan structural alteration in cells

The alteration of glycosyltransferase expression and the subsequent changes in oligosaccharide structures are reported in several diseases. The analysis of glycan structural alteration in glycoproteins is becoming increasingly important in the discovery of therapies and diagnostic markers. In this study, we propose a strategy for glycomic/glycoproteomic analysis based on oligosaccharide profiling by LC/MS followed by proteomic approaches, including 2-DE and 2-D lectin blot. As a model of aberrant cells, we used Chinese hamster ovary cells transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to beta-mannose of the mannosyl core of N-linked oligosaccharides. LC/MS equipped with a graphitized carbon column (GCC) enabled us to elucidate the structural alteration induced by the GnT-III expression. Using 2-D lectin blot followed by LC/MS/MS, the protein carrying an extra N-acetylhexosamine in cells transfected with GnT-III was successfully identified as integrin alpha3. Thus, oligosaccharide profiling by GCC-LC/MS followed by proteomic methods can be a powerful tool for glycomic/glycoproteomic analysis.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Identification of spin trapped carbon-centered radicals in soybean lipoxygenase-dependent peroxidations of omega-3 polyunsaturated fatty acids by LC/ESR,LC/MS,and tandem MS

With the combined techniques of on-line liquid chromatography/electron spin resonance (LC/ESR) and on-line liquid chromatography/mass spectrometry (LC/MS), we have previously characterized all classes of lipid-derived carbon-centered radicals (*Ld) formed from omega-6 polyunsaturated fatty acids (PUFAs: linoleic acid and arachidonic acid). In the present study, the carbon-centered radicals formed from two omega-3 PUFAs (linolenic acid and docosahexaenoic acid) resulting from their reactions with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butylnitrone (POBN) were investigated using the combination of LC/ESR and LC/MS techniques. A total of 16 POBN trapped carbon-centered radicals formed from the peroxidation of linolenic acid and 11 formed from the peroxidation of docosahexaenoic acid were detected by LC/ESR, identified by LC/MS, and structurally confirmed by tandem mass analysis (MS/MS). The on-line ESR chromatograms and MS chromatograms obtained from two omega-3 PUFAs closely resembled each other not only because the four major beta-scission products, including an ethyl radical and three isomeric pentenyl radicals, were formed from each PUFA, but also because isomeric POBN adducts of lipid dihydroxyallylic radicals from both PUFAs had almost identical chromatographic retention times.     

Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography

A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preoperatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.     

Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry.

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (< or = 25) picomole level during chromatography of glycoprotein digests and for differentiation of O-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from O-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three O-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.     

Densimetric determination of carbohydrate content in glycoproteins.

Carbohydrates play important roles in activity, stability and pharmacokinetics of glycoproteins and the degree of glycosylation varies with proteins. In this communication, a simple method of determining the carbohydrate content was developed, which consists of measuring the density increments of a glycoprotein and its non-glycosylated counterpart, and then dividing the difference between the two values by the density increment of carbohydrates. The density increment was relatively constant for various sugars except for sialic acid, and hence assumed to be 0.39. Thus, we obtained carbohydrate contents of 38, 28, 8 and 7% for Chinese hamster ovary cell-expressed erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and platelet-derived growth factor (PDGF), respectively. These values are in close agreement with those determined by other methods.     

Ketosynthase Domain Probes Identify Two Subclasses of Fungal Polyketide Synthase Genes

Analysis of fungal polyketide synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketosynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketosynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal polyketide synthase genes.     

Secretion of a lactosaminoglycan-containing glycoprotein by peri-implantation sheep conceptuses

Sheep conceptuses from day 16 of pregnancy were cultured in the presence of [3H]glucosamine and [14C]leucine and a high-molecular-weight glycoprotein (HMWG) secreted into the culture medium was purified by a combination of anion-exchange and gel filtration chromatography. The HMWG was found to have a molecular weight between 800,000 and 900,000 and to be highly resistant to digestion with pronase. Characteristics of the carbohydrate portion of the purified glycoprotein were examined by selective chemical and enzymatic digestions and lectin binding studies. Mild alkaline reduction was ineffective in disassociating carbohydrate chains from the protein core. Furthermore, the protein was resistant to both O-glycanase and peptide:N-glycanase F. Harsh alkaline reduction caused the release of carbohydrates, however. After pronase digestion of these products, three molecular weight classes of carbohydrates were resolved by Sephadex G-25 chromatography. Two lines of evidence indicate that the HMWG contains lactosaminoglycan components. The intact molecule and two of the molecular weight classes of carbohydrates resolved by harsh alkaline reduction bind Datura stramonium lectin. Binding of HMWG to lectin could be partially inhibited by N-acetyllactosamine and completely inhibited by a mixture of N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. Secondly, digestion with endo-beta-galactosidase causes the release of 16% of the [3H]glucosamine from the intact molecule. Therefore, the HMWG of the sheep conceptus is the first reported example of secretion of lactosaminoglycan-containing glycoprotein by peri-implantation embryos.     

The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.