Analysis of glycoprotein heterogeneity by capillary electrophoresis and mass spectrometry

Glycosylation is a complex posttranslational modification that can result in extensive heterogeneity for recombinant glycoproteins produced by eukaryotic systems. The carbohydrate moiety of a recombinant glycoprotein may affect the immunogenicity, half-life, bioactivity, and stability of a potential therapeutic product. Regulatory authorities such as the US Food and Drug Administration demand increasingly sophisticated carbohydrate analysis to ensure product characterization, batch-to-batch consistency, and stability. The advent of new technologies for analysis of biopolymers by capillary electrophoresis and mass spectrometry has revolutionized strategies for recombinant protein characterization. In particular, recent advances in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry now permit relatively rapid and detaned assessment of glycoprotein and oligosaccharide structure. In this article, we describe some applications of capillary electrophoresis and mass spectrometry to monitor the glycosylation associated with a model recombinant glycoprotein, human interferon-γ.     

Protein glycosylation analysis with capillary-based electromigrative separation techniques

An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-of-principle studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.     

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.     

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.     

CRSBP-1/LYVE-l-null mice exhibit identifiable morphological and functional alterations of lymphatic capillary vessels

CRSBP-1, a membrane glycoprotein, can mediate cell-surface retention of secreted growth factors containing CRS motifs such as PDGF-BB. CRSBP-1 has recently been found to be identical to LYVE-1, a specific marker for lymphatic capillary endothelial cells. The in vivo role of CRSBP-1/LYVE-1 is unknown. CRSBP-1-null mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic capillary vessels in certain tissues, marked by the constitutively increased interstitial-lymphatic flow and lack of typical irregularly-shaped lumens. The CRSBP-1 ligands PDGF-BB and HA enhance interstitial-lymphatic flow in wild-type mice but not in CRSBP-1-null animals.     

Purification and characterization of a soluble glycoprotein from garlic (Allium sativum) and its in vitro bioactivity

A soluble glycoprotein was purified to homogeneity from ripe garlic (Allium sativum) bulbs using ammonium sulfate precipitation, Sephadex G-100 gel filtration, and diethylaminoethyl-52 cellulose anion-exchange chromatography. A native mass of 55.7?kDa estimated on gel permeation chromatography and a molecular weight of 13.2?kDa observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported that the glycoprotein is a homotetramer. β-Elimination reaction result suggested that the glycoprotein is an N-linked type. Fourier-transform infrared spectroscopy proved that it contains sugar. Gas chromatography–mass spectrometer analysis showed that its sugar component was galactose. The glycoprotein has 1,1-diphenyl-2-picrylhydrazil free radical scavenging activity and the peroxidation inhibition ability to polyunsaturated fatty acid. These results indicated that the glycoprotein has potential for food additives, functional foods, and even biotechnological and medical applications.     

Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis.

A quick and reproducible capillary electrophoresis method was optimized and validated for the assay of bovine serum albumin (BSA). The effects of various parameters such as pH of buffer, concentration of buffer, capillary dimensions, use of coated capillaries, and additives such as surfactants and protein solubilizers were evaluated. The capillary coatings or additives did not give any advantage in reducing the surface adsorption of BSA on the capillary walls. The optimized conditions include use of borate buffer, pH 8.5 having a concentration of 150 mM in a 27 cm capillary with an aperture window of 100 x 200 microns for detection. The optimized method for the detection of BSA was validated. The interday and intraday coefficient of variation was not greater than 7.59% at BSA concentrations of 25-1000 micrograms/ml. The method developed was reproducible and accurate.     

Separation of proteolytic enzymes originating from Antarctic krill (Euphausia superba) by capillary electrophoresis

Extracts prepared from Antarctic krill (Euphausia superba), mainly consisting of acidic proteolytic enzymes, have been studied with capillary electrophoretic techniques. Approximately 50 repeatable peaks were obtained with capillary zone electrophoresis on an untreated fused-silica capillary using a phosphate buffer containing anionic and cationic fluorosurfactant additives as separation medium. A faster separation was achieved on a polyvinyl alcohol coated capillary. Quantitative variations of individual proteins regarding different krill enzyme batches were noted. In the krill samples trypsin-like serine proteinase, carboxypeptidase A and carboxypeptidase B were tentatively identified.     

Evaluation of the use of cyclodextrins in chiral separation of basic drug substances by capillary electrophoresis

The influence of the choice of type and/or concentration of cyclodextrin, other additives, the temperature surrounding the capillary, and buffer pH on the separation of some chiral basic drug substances in capillary zone electrophoresis has been evaluated. It was found that pH of the buffer and type and concentration of cyclodextrin had a major influence on the separation. © 1995 Wiley-Liss, Inc.     

Enantiomer separation by capillary electrophoresis utilizing noncyclic mono-, oligo- and polysaccharides as chiral selectors

Various noncyclic mono-, oligo- and polysaccharides have been successfully used for enantiomer separation in the analytical sciences such as HPLC and capillary electrophoresis (CE). This review presents enantiomer separation by CE utilizing mainly polysaccharides as chiral additives. The operation conditions that affect the enantioselectivity are briefly discussed.     

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.     

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.     

Ultramicroanalysis of reducing carbohydrates by capillary electrophoresis with laser-induced fluorescence detection as 7-nitro-2,1,3-benzoxadiazole-tagged N-methylglycamine derivatives

A method for ultramicroanalysis of carbohydrates using capillary electrophoresis with laser-induced fluorescence detection was developed, based on precapillary conversion to 7-nitro-2,1, 3-benzoxadiazole (NBD)-tagged N-methylglycamines. Although the derivatization involves two-step reactions, i.e., reductive N-methylamination followed by condensation with NBD-F, they can be carried out in a one-pot fashion and proceed quantitatively within ca. 50 min in total. Since the reaction conditions are mild, it does not cause desialylation. The derivatives can be well separated by capillary electrophoresis and sensitively detected by argon laser-induced fluorescence. It allowed detection of monosaccharides of down to nanomolar concentrations for analytical sample solution, which correspond to the attomole injected amounts, and good linearity was observed over a wide range. It was also successfully applied to analysis of N-glycans in a microgram quantity of a glycoprotein. Studies on the cleanup of derivatized product are also described in relation to N-glycan analysis.     

Determination of site-specific glycan heterogeneity on glycoproteins

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.     

Nephritogenoside,the receptor glycoprotein for concanavalin a in rat glomerular basement membrane: Demonstration of α-glucopyranosyl unit at the non-reducing terminus

Rat glomerular basement membrane was extracted for 3 h with trypsin, pH 8.0. The supernatant solution was treated with trichloroacetic acid and the supernatant thus obtained was applied to Bio-Gel P200. The first of the two glycoprotein peaks was applied onto Sepharose derivatives of concanavalin A (Con A.).Examination of the material retained by the unsolubilized Con A and subsquently eluted with methyl α-d-mannopyranoside reveals that the principal high affinity receptor for Con A is the renal glycoprotein, having antigenic activity that induces nephrotoxic antibody. This glycoprotein has also nephritogenicity (the activity capable of inducing glomerulonephritis in homologous animals by a single foot pad injection with Freund's incomplete adjuvant). Evidence is given to show that this binding is specific.The remainder of the renal glycoprotein is unretarded and is revealed to contain none of the activities described above.When fluorescein isothiocyanate-labelled Con A is, conversely, injected into rats through the renal artery, the specific binding of Con A to the glomerular capillary loop is proved.The results demonstrated here appear to, indicate that the receptor for Con A present in normal rat glomerular basement membrane can be identified as the well-established chemical substance, the nephritogenoside, having the α-d-glucopyranosyl unit at the non-reducing terminus which is facing the endothelial aspects of the glomerular capillary loop.     

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.     

The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type i by capillary zone electrophoresis

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.Abbreviations CDGS Carbohydrate Deficient Glycoprotein Syndrome - CZE Capillary Zone Electrophoresis - hTf human transferrin - gu HPLC glucose units - EOF electroosmotic flow. Nomenclature: for describing oligosaccharide structures: A(1,2,3,4) indicates the number of antennae linked to the t trimannosyl core - G(0–4) indicates the number of terminal galactose residues in the structure - F core fucose - B bisecting N-acetyl glucosamine (GlcNAc) - S sialic acid - Gal galactose; M - Man mannose     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

Plasma membrane glycoproteins of tumour cells in enzootic bovine leukosis compared with normal bovine lymphoid cells

Plasma membranes from tumor cells in enzootic bovine leukosis and from normal bovine lymphoid cells of different sources have been isolated and characterized. The glycoprotein composition of the different membranes has been studied by SDS-PAGE and by analysis of the carbohydrate composition as well as by lectin binding of glycoprotein fractions obtained by phenol treatment of the membranes. No differences in the electrophoretical glycoprotein pattern could be detected comparing (nonmalignant) cells from persistent lymphocytosis and tumour cells, suggesting that malignancy is not associated with the gain or loss of a major glycoprotein. A 151.5 kD glycoprotein, present in membranes from normal lymph node lymphocytes, seems to be absent in the membranes of peripheral blood lymphocytes as well as of tumour cells. The loss of this glycoprotein might thus be associated with a loss of sessility of bovine lymphoid cells. The carbohydrate analysis and lectin binding of extracted glycoproteins revealed a decreased fucosylation accompanied by an increased exposure of galactose residues as well as a loss or a decreased complexity of N-glycosidically bound oligosaccharides in the tumour cell glycoproteins compared with those of normal cells. These findings are discussed with regard to disturbed growth regulation of leukosis tumour cells.