Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCC-LC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d4-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d6-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with d0- and d6-AP, respectively, and an equal amount of d0- and d4-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases.     

Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals

Carbohydrate chains in glycoprotein pharmaceuticals play important roles for the expression of their biological activities, but the structure and compositions of carbohydrate chains are dependent on the conditions for their production. Therefore, evaluation of the carbohydrate chains is quite important for productive process development, characterization of product for approval application, and routine quality control. The oligosaccharides themselves have complex structure including blanching and various glycosidic linkages, and oligosaccharides in one glycoprotein pharmaceutical generally have high heterogeneity, and characterization of oligosaccharide moiety in glycoprotein has been a challenging target. In these situations, CE has been realized as a powerful tool for oligosaccharide analysis due to its high resolution and automatic operating system. This review focuses on the application of CE to the glycoform analysis of glycoproteins and profiling of the N-linked glycans released from glycoprotein pharmaceuticals. Current applications for structure analysis using CE-MS(n) technique and glycan profiling method for therapeutic antibody are also described.     

Simultaneous microanalysis of N-linked oligosaccharides in a glycoprotein using microbore graphitized carbon column liquid chromatography-mass spectrometry

We previously reported that graphitized carbon column liquid chromatography-mass spectrometry (GCC-LC-MS) is very useful for the structural analysis of carbohydrates in a glycoprotein. In this study, GCC-LC-MS was adapted for the simultaneous microanalysis of oligosaccharides. A variety of oligosaccharide alditols prepared from fetuin, ribonuclease B, and recombinant human erythropoietin were used as model oligosaccharides. The use of microbore GCC-LC-MS was found to be successful for rapid, sensitive, and simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols. Furthermore, we demonstrate that this method is applicable to the analysis of carbohydrate heterogeneity in a glycoprotein that possesses diverse oligosaccharides. Microbore GCC-LC-MS was able to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator produced from human melanoma cells in a single analysis.     

Exploration of oligosaccharide-protein interactions in glycoprotein quality control by synthetic approaches

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides, play important roles in glycoprotein quality control. This process is highly complex, involving a number of lectins, chaperones, and glycan-processing enzymes. For example, calnexin and calreticulin (CRT) are molecular chaperones that recognize monoglucosylated forms of high-mannose-type glycans. UDP-glucose : glycoprotein glucosyltransferase (UGGT) only glucosylates high-mannose-type glycans attached to partially folded proteins. Fbs1 is a component of ubiquitin ligase that recognizes sugar chains. Although recent studies have clarified the properties of these proteins, most of them used oligosaccharides derived from natural sources, which contain structural heterogeneity. In order to gain a more precise understanding, we started our program to comprehensively synthesize high-mannose-type glycans associated with a protein quality control system. Additionally, investigation of artificial glycoproteins led us to the discovery of the first nonpeptidic substrate of UGGT. These synthetic oligosaccharide probes have allowed us to conduct quantitative evaluations of the activity and specificity of CRT, Fbs1, and UGGT.     

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.     

Rapid analysis of oligosaccharides derived from glycoproteins by microchip electrophoresis

A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (mu-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G2), cellobiose ( G2'), maltriose (G3) and panose (G3') as oligosaccharide isomer models. In mu-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, alpha1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by mu-CE, indicating that the present mu-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.     

Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high-performance anion-exchange chromatography and nuclear magnetic resonance spectroscopy.

The characterisation of oligosaccharides present on albumin Casebrook, a glycoprotein variant of human serum albumin, which contains an N-linked oligosaccharide at an attachment site formed by a point mutation of 494 Asp-->Asn, is described. The monosaccharide compositional analysis of purified glycopeptides suggested the presence of complex biantennary carbohydrate structures. The oligosaccharides which were released by N-glycosidase-F appeared to be a single molecular species according to their retention on high-performance anion-exchange chromatography. The structure of the oligosaccharide was suggested by sequential exoglycosidase digestions and confirmed by proton nuclear magnetic resonance spectroscopy. It was concluded that the oligosaccharides were essentially homogeneous and consisted of an alpha(2-6)-desialylated complex biantennary glycan.     

Fluorous-Assisted One-Pot Oligosaccharide Synthesis

A new method for oligosaccharide assembly that combines the advantages of one-pot synthesis and fluorous separation is described. After one-pot glycosylations are completed, a fluorous tag is introduced into the reaction mixture to selectively "catch" the desired oligosaccharide, which is rapidly separated from non-fluorous impurities by fluorous solid-phase extraction (F-SPE). Subsequent "release" of the fluo rous tag and F-SPE achieved the purification of the desired oligosaccharide without the use of time- and solvent-consuming silica gel chromatography. Linear and branched oligosaccharides have been synthesized with this approach in just a few hours (for the overall oligosaccharide assembly and purification process).     

Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry

A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.     

Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies

Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-aminobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency.     

Cap and capture-release techniques applied to solid-phase synthesis of oligosaccharides

This paper reports a new strategy for oligosaccharide synthesis by combining solid-phase methods with cap and capture-release separation techniques, using the p-(5-(ethoxycarbonyl)pentyloxy)benzyl group (CPB) as a tag for the capture of desired oligosaccharides. After a complex carbohydrate mixture was obtained by solid-phase synthesis, the desired oligosaccharide containing a free carboxyl group derived from CPB was attached to an amino resin. The loaded resin was readily separated from side products by filtration and finally treated with acid to release the pure oligosaccharide product.     

Advances in Selective Chemical Syntheses of Complex Oligosaccharides

A wide variety of complex oligosaccharides has now been made accessible as a result of methodological improvements in the sphere of chemical synthesis, which can be used for the study of conformations and interactions with protein-receptor molecules. Such work is of particular importance because the oligosaccharide chains of glycoproteins and glycolipids anchored to the plasma membrane are important in cell-cell interactions and are receptors for enzymes, hormones, proteins, and viruses; furthermore, they determine the antigen properties of cells. The methods of synthesis available for the production of selective linkages in oligosaccharides are dealt with, particular attention being paid to conversion of saccharides into oligosaccharides, which are important from the biological point of view.     

Rapid analysis of N‐linked oligosaccharides in glycoproteins (ovalbumin,ribonuclease B and fetuin) by reversed‐phase ultra‐performance liquid chromatography with fluorescence detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid, selective and sensitive determination of N‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra‐performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). The asparaginyl‐oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel‐permeation chromatography were labeled with 1‐pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF‐MS. The PSC‐labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI‐TOF‐MS. As the results, 15, eight and four kinds of N‐linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N‐linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd.     

The Non-degradative Isolation of α1-Acid Glycoprotein from Normal and Rheumatoid Plasma1

Abstract

We have developed a method for the purification of α₁-acid glycoprotein (AGP) using procedures unlikely to damage the glycoprotein structure. This was utilised to isolate AGP from samples of normal and rheumatoid plasma. The effectiveness of the purification procedure was examined by enzymatically deglycosylating each sample of AGP, separating the released oligosaccharides by chromatography on a pellicular high pH anion-exchange (HPAE) resin at pH 13 and detecting by a pulsed electrochemical (PED) method. The analytical profile for normal AGP was consistent with those previously reported thus indicating that the purification procedure did not denature the oligosaccharide chains of AGP; there was a noticeable difference between AGP in normal and rheumatoid plasma.     

Oligosaccharide synthesis and library assembly by one-pot sequential glycosylation strategy

The oligosaccharide residue in glycoconjugates located in cell membranes is responsible for intercellular recognition and interaction: it acts as a receptor for proteins, hormones, and microorganisms and governs immune reactions. These significant activities have stimulated great interest in the field of oligosaccharides and glycoconjugates. Although many advances have been made in the synthesis of oligosaccharides, more convenient and efficient methods are still needed. This review describes one of these new methods-the one-pot sequential glycosylation approach as a potent tool for oligosaccharide assembly. The oligosaccharide library construction in a one-pot fashion is also summarized.     

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的-NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究.     

Applications of isotopes in advancing structural and functional heparanomics

Heparanomics is the study of all the biologically active oligosaccharide domain structures in the entire heparanome and the nature of the interactions among these domains and their protein ligands. Structural elucidation of heparan sulfate and heparin oligosaccharides is a major obstacle in advancing structure–function relationships and heparanomics. There are several factors that exacerbate the challenges involved in the structural elucidation of heparin and heparan sulfate; therefore, there is great interest in developing novel strategies and analytical tools to overcome the barriers in decoding the enigmatic heparanome. This review focuses on the applications of isotopes, both radioisotopes and stable isotopes, in the structural elucidation of the complex heparanome at the disaccharide or oligosaccharide level using liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. This review also outlines the utility of isotopes in determining the substrate specificity of biosynthetic enzymes that eventually dictate the emergence of biologically active oligosaccharides.     

Structural characterization by chromatographic profiling of the oligosaccharides of human immunodeficiency virus (HIV) recombinant envelope glycoprotein gp120 produced in Chinese hamster ovary cells

This report together with the paper by T. Mizuochi, M. W. Spellman, M. Larkin, J. Solomon, L. J. Basa and T. Feizi (1988) Biochem. J. 254, 599-603 describes the structural elucidation of the N-linked oligosaccharides of the HIV envelope glycoprotein, gp120 (cloned from the HTLV-III B isolate and expressed as a secreted fusion protein after transfection of Chinese hamster ovary cells), which is known to bind with high affinity to human T4 lymphocytes. Oligosaccharides were released from peptide by hydrazinolysis, fractionated by paper electrophoresis, high performance lectin affinity chromatography and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. The glycoprotein was found to be unique in its diversity of oligosaccharide structures. These include high-mannose type and hybrid type, as well as four categories of complex type chains: mono-, bi-, tri- and tetra-antennary, with or without N-acetyllactosamine repeats, and with or without a core region fucose residue. Among the sialidase-treated oligosaccharides no less than 29 structures were identified as follows: (formula; see text) where G = galactose; GN = N-acetylglucosamine; M = mannose; F = fucose; +/- = residues present in a proportion of chains. The actual number of oligosaccharide structures is much greater since before desialylation there was evidence that among the hybrid and complex type chains all but 6% contained sialic acid at the C-3 position of terminal galactose residues, and partially sialylated forms of the bi- and multiantennary chains were present.     

Preparative purification of tyrosinamide N-linked oligosaccharides

N-linked oligosaccharides from glycoproteins can be either analyzed on a sub-nanomole scale or preparatively purified on a multi-micromole scale. Each goal necessitates a unique analytical strategy often involving oligosaccharide derivatization to enhance separation and detection. Tyrosinamide-oligosaccharides were developed to facilitate the preparative purification of N-linked oligosaccharides. These have found many uses in oligosaccharide remodeling, in the preparation of neoglycoconjugates, in developing receptor probes, and even as analytical standards in chromatography. This review discusses progress in the preparation of tyrosinamide-oligosaccharides from different glycoproteins and their utility in glycobiology research.     

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.