Subject(s)
Chromatography, High Pressure Liquid/methods , Glycoproteins/analysis , Animals , CHO Cells , Cricetinae , Fluorescent Dyes/metabolism , Glycoproteins/chemistry , Humans , Immunoglobulin G/chemistry , Models, Chemical , Monosaccharides/analysis , Sialic Acids/analysis , Sialic Acids/metabolism , Swine , Time FactorsABSTRACT
Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose.
Subject(s)
Carbohydrates/analysis , Electrophoresis, Polyacrylamide Gel/methods , Fluorescent Dyes/chemistry , ortho-Aminobenzoates/chemistry , Animals , CHO Cells , Chromatography, High Pressure Liquid , Cricetinae , Glycoside Hydrolases/chemistryABSTRACT
Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.
Subject(s)
Sequence Analysis/instrumentation , Immunoglobulin G/chemistry , Lactoglobulins/chemistry , Software , Time FactorsABSTRACT
A capillary isoelectric focusing (cIEF) method was developed for routine analysis of recombinant immunoglobulins (rlgGs). The cIEF method used a dimethyl siloxane-coated capillary and a separation matrix of 2% ampholytes in 0.4% methylcellulose (MC). The rIgGs, and internal pI marker protein standards, were mixed with carrier ampholyte in MC, focused using high voltage, and then the protein bands were mobilized past a UV detector by simultaneous application of low pressure and voltage. Qualitatively and quantitatively equivalent rIgG focusing profiles were obtained via cIEF and gel-based IEF, with individual isoform peak area percentages and calculated peak pI values being comparable for the same samples. Linear relationships were obtained for peak area response versus sample concentration, and for the pI gradient developed between the internal pI marker standards. The relative standard deviation (RSD) in rIgG peak areas was less than 2% intra-day and less than 8% inter-day (72 h). The RSD for the mobilization times of rIgG peaks was less than 1% intra-day and less than 3% inter-day (72 h). There was no observed decrease in the performance of the capillary over 150 analyses. cIEF offers several important advantages over gel IEF, e.g. direct, quantitative detection of proteins by intrinsic UV absorbance at 280 nm, rapid analyses ( < or = 30 min), capability of automation, and one-step, electronic data analysis and archival. These data demonstrate the superiority of the cIEF method for routine analysis of rIgGs.
Subject(s)
Immunoglobulins/analysis , Protein Isoforms/analysis , Recombinant Proteins/analysis , Buffers , Calibration , Evaluation Studies as Topic , Hydrogen-Ion Concentration , Isoelectric Focusing/instrumentation , Isoelectric Focusing/methods , Reproducibility of Results , Siloxanes/chemistry , Time FactorsABSTRACT
We developed a novel chemistry for C-terminal sequencing of proteins based on derivatization with acetylisothiocyanate to yield amino acid thiohydantoins (TH-AAs), and it was used for manual sequencing of biopharmaceutical products on a routine basis.This simple chemistry was automated using a ABI 473A N-terminal sequencer. All reagents (R1, trimethylsilylisothiocyanate; R3, alkaline thiocyanate for cleavage) and solvents required for sequencing were accommodated on the sequencer, which was modified to deliver liquid R2 (acetyl chloride) to the reaction vessel.The conversion flask was used for preparing the TH-AAs for analysis by online HPLC using a graphitized carbon (Hypercarb) column. Results obtained with model proteins and recombinant protein drugs suggest that at least three residues from the C terminus can be easily determined.The C-terminal heterogeneity in more than five types of recombinant immunoglobulin G was determined, and the differences in Gly-Lys ratios were consistent with changes observed in the isoelectric focusing profile of these antibodies. Because the chemistry uses only four reagents delivered to the reaction vessel and three to the conversion flask,we believe that the automated protocol can be easily adapted to any existing N-terminal sequencer.
ABSTRACT
Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.
Subject(s)
Glycoproteins/chemistry , Oligosaccharides/chemistry , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, High Pressure Liquid/methods , Humans , Molecular Sequence Data , Oligosaccharides/isolation & purification , Sensitivity and Specificity , Spectrometry, Fluorescence/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , ortho-AminobenzoatesABSTRACT
The relatively rapid and extensive characterization of the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncytial virus (RSHZ19) is presented. The integrated strategy used a combination of mass spectrometric and conventional methodologies. Liquid chromatography/electrospray mass spectrometry was used for peptide mapping and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of selected peptides. Matrix-assisted laser desorption/ionization mass spectrometry provided the M(r) of the intact protein and was used to characterize endo- and exoglycosidase digests of isolated glycopeptides to identify the glycosylation-site peptide and define the structures of the carbohydrates at that site. These experiments verified 99.1% of the light- and 99.3% of the heavy-chain amino acid sequences. The N and C termini of both chains were confirmed, and the nature and extent of heterogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demonstrated by sequencing the N-terminally blocked peptide by tandem MS. Carbohydrate was found exclusively at Asn296 of the heavy chain. There was no evidence for a nonglycosylated form of the molecule or for the presence of O-linked carbohydrate. The qualitative distribution of glycoforms at this site was determined by MS of the isolated, tryptic glycopeptide and compared with results obtained by high-performance anion exchange chromatography and high-resolution gel permeation chromatography of oligosaccharides released by hydrazinolysis. The sequence and linkage of individual glycan species were determined using matrix-assisted laser desorption/ionization MS to monitor the results of a series of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated carbohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consistency of the MS methods with conventional methods for carbohydrate analysis.
Subject(s)
Antibodies, Monoclonal/chemistry , Carbohydrates/chemistry , Proteins/chemistry , Respiratory Syncytial Viruses/immunology , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal, Humanized , Carbohydrates/analysis , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Humans , Molecular Sequence Data , Proteins/analysis , Respiratory Syncytial Viruses/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Sialic acids were specifically labeled with o-phenylenediamine-2HCl (OPD) to yield stable fluorescent quinoxaline derivatives. The sialic acids were released from the glycoprotein in a NaHSO4 solution (0.25 M 80 degrees C, 20 min) and derivatized in the same solution with the OPD (10 mg/ml (final conc.) 80 degrees C, 40 min). Various sialic acids derivatized with the OPD were separated on a C-18 reversed-phase Ultrasphere-ODS column using the solvent systems and the detector conditions used for the determination of monosaccharides derivatized with anthranilic acid as reported earlier. The common N-acetyl- and N-glycolylneuraminic acids were separated within 20 min, and the other N- and O-acylated sialic acids were separated in 40 min. The OPD derivatives of mono-, di-, and triacylated sialic acid were separated into their respective groups in the present separation. The fluorescence maxima for the OPD-N-acetylneuraminic acid were 232 nm excitation and 420 nm emission and the limit of quantitation was < 2 pmol. The relative standard deviation was less than 3.0% for the sialic acid determinations using the glycoproteins. A common single HPLC system is used for complete carbohydrate composition analysis of glycoproteins, since both the sialic acid and the monosaccharide methods use the same solvent systems, column, and detector settings. Furthermore, in contrast to high-performance anion-exchange chromatography with pulsed amperometric detection, these methods are easy to set up for an analysis and offer the highest sensitivity for analyzing samples available in microgram amounts.
Subject(s)
Chromatography, High Pressure Liquid/methods , Glycoproteins/analysis , Sialic Acids/chemistry , Linear Models , Phenylenediamines , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Fluorescence , Time FactorsABSTRACT
A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.
Subject(s)
Chromatography, High Pressure Liquid/methods , E-Selectin/chemistry , Amino Acids/analysis , Animals , CHO Cells , Carbohydrates/analysis , Cell Adhesion , Cell Line , Cricetinae , E-Selectin/isolation & purification , E-Selectin/physiology , HL-60 Cells , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, UltravioletABSTRACT
We have isolated and sequenced a Neurospora crassa gene encoding a single copy of ubiquitin (UBI) fused to the S27a ribosomal (r) protein. We have opted to name this gene the ubiquitin/cytoplasmic r-protein gene 6 (ubi::crp-6). The ubi::crp-6 gene generates a 700-nucleotide (nt) transcript. It shares a 700-bp regulatory region with the cytoplasmic r-protein gene 5 (crp-5), a gene encoding the N. crassa S26 r-protein. The two genes are transcribed divergently from the common regulatory region and their mRNA levels are regulated in parallel during growth on a variety of carbon sources.
Subject(s)
Genes, Fungal , Neurospora crassa/genetics , Ribosomal Proteins/genetics , Ubiquitins/genetics , Amino Acid Sequence , Base Sequence , DNA, Fungal/genetics , Gene Expression Regulation, Fungal , Molecular Sequence Data , RNA, Messenger/genetics , Restriction Mapping , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
For specific determination of monosaccharides with high sensitivity, glycoprotein acid hydrolysates were derivatized in a simple step with excess anthranilic acid (2-aminobenzoic acid) in the presence of sodium cyanoborohydride to give highly fluorescent stable derivatives. The monosaccharide derivatives were completely separated from the excess reagent and from each other by HPLC on a C-18 reversed-phase column using a 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase. Reductive amination of the monosaccharides in the methanol-acetate-borate medium was complete within 20 min at 80 degrees C. Derivatization of glucosamine with the anthranilic acid was accompanied by epimerization to mannosamine (> 15%) in methanol-acetic acid reaction medium, but it was reduced to < 3% in methanol-acetate-borate reaction medium. Fluorescence intensity of the hexosamines was greater than twice the intensity of the neutral monosaccharides. The fluorescent derivatives had excitation maxima at 230, 245, and 360 nm and an emission maximum at 425 nm. Fluorescence intensity at 230 nm excitation was about 10 times greater than that obtained with excitation at 360 nm for all the monosaccharides. Release and concomitant destruction of the monosaccharides during hydrolysis in 20% TFA at 100 degrees C for 7-8 h resulted in 83-85% recovery of all the monosaccharides from glycoproteins. The monosaccharide compositions determined by this method were in excellent agreement with the expected values for a recombinant immunoglobulin and fetuin and were highly reproducible. Relative standard deviation for the composition determinations and precision was less than 3%.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Glycoproteins/chemistry , Hexosamines/analysis , Monosaccharides/analysis , Animals , CHO Cells , Chromatography, High Pressure Liquid/methods , Cricetinae , Hydrolysis , Indicators and Reagents , Sensitivity and Specificity , Spectrometry, Fluorescence/methods , ortho-AminobenzoatesABSTRACT
Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.
Subject(s)
Procollagen/biosynthesis , Amino Acid Sequence , Amino Acids/analysis , Chromatography, DEAE-Cellulose , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Procollagen/chemistry , Procollagen/genetics , Procollagen/isolation & purification , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transfection , Tumor Cells, CulturedABSTRACT
Endo beta-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N'-diacetylchitobiose, N,N'-diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the endo beta-N-acetylglucosaminidase F (Endo F) activity on various free oligosaccharides obtained from the standard glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase) mixture readily cleaved high mannose and complex oligosaccharides (neutral and sialyated) with common core alpha 1-6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core alpha 1-6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/metabolism , Oligosaccharides/metabolism , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Fucose/chemistry , Fucose/metabolism , Humans , In Vitro Techniques , Mannose/chemistry , Mannose/metabolism , Molecular Sequence Data , Oligosaccharides/chemistry , Orosomucoid/chemistry , Orosomucoid/metabolism , Substrate SpecificityABSTRACT
Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivatives were prepared by a one-step reaction with N-methylisatoic anhydride (MIA) for quantitative detection in HPLC. Reactions were carried out in an organic medium of acetonitrile-triethylamine, in aqueous alkaline sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the organic reaction medium. The DMAP had no effect in the aqueous buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin had excitation and fluorescence maxima at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixtures, respectively. Fluorescence intensity increased with an increase in ACN concentration and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivatives obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was about 50-100 times more sensitive (lower limits of 0.1 to 0.5 picomole) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.
Subject(s)
4-Aminopyridine/analogs & derivatives , Anhydrides , Fluorescent Dyes/chemistry , Kinins/chemistry , Peptides/chemistry , ortho-Aminobenzoates/chemistry , Bradykinin/chemistry , Chromatography, High Pressure Liquid , Kinins/analysis , Peptides/analysis , Solvents , Spectrometry, Fluorescence , WaterABSTRACT
Various steps involved in the preparation of partially methylated alditol acetates (PMAAs) from glycoprotein-derived carbohydrates were improved to obtain the derivatives in a rapid manner with excellent yields. Carbohydrates were permethylated in dimethyl sulfoxide (DMSO), using a fine suspension of sodium hydroxide and methyl iodide (CH3I). The fine suspension of NaOH was prepared conveniently from commercially available 50% aqueous NaOH in DMSO by sonication and washing the precipitate with DMSO. Methylation of ovalbumin and fetuin glycopeptides using the fine suspension of NaOH and CH3I was complete within 5 min, and the methylation reaction did not generate any nonsugar artifacts. Methylated carbohydrates without any purification were hydrolyzed in a mixture of volatile organic acids, which permitted rapid removal of the acids from samples by evaporation. Acetylation of partially methylated alditols with acetic anhydride for 2-4 h at ambient temperature using 4-N,N'-dimethylaminopyridine as a catalyst and the reaction was free from generating nonsugar reaction artifacts. The reaction time course for methylation, hydrolysis, and acetylation was determined to obtain optimum reaction conditions for preparation of the PMAAs. The procedure facilitated rapid identification and quantitation of PMAAs due to diminished reaction artifacts and the quality of the chromatogram depended only on the purity of starting material and the reagents used for the methylation analysis. Utility of these simple methods for rapid methylation analysis was demonstrated in the characterization of oligosaccharides isolated in small amounts using a carbohydrate analyzer.
Subject(s)
Acetates/isolation & purification , Carbohydrates/chemistry , Glycoproteins/chemistry , Sugar Alcohols/isolation & purification , Acetates/chemistry , Acetylation , Animals , Chromatography, Gas , Dimethyl Sulfoxide , Hydrolysis , Kinetics , Methylation , Oxidation-Reduction , Sugar Alcohols/chemistryABSTRACT
Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.
Subject(s)
Amino Sugars/analysis , Chromatography, High Pressure Liquid/methods , Sugar Alcohols/analysis , Galactosamine/analysis , Glucosamine/analysis , Glycoproteins/chemistry , Isothiocyanates , ThiocyanatesABSTRACT
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.
Subject(s)
CD4 Antigens/analysis , Glycoproteins/chemistry , Oligosaccharides/chemistry , Ovary/immunology , Amino Acid Sequence , Animals , Binding Sites , CD4 Antigens/immunology , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cricetinae , Cricetulus , Female , Glycoproteins/immunology , Methylation , Molecular Sequence Data , Oligosaccharides/classification , Ovary/cytology , Solubility , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide N-glycosidase F. endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion-exchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1-0.3 microA output with suitable sensitivity (less than 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients. Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestion (greater than 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ion-exchange chromatography of the acqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and alpha 1-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri- and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with alpha-mannosidase. Treatment with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with alpha-L-fucosidase, endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue. Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo- and exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.
Subject(s)
Asparagine , Glycoproteins/chemistry , Oligosaccharides/chemistry , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Ion Exchange , Glycoside Hydrolases , Humans , Indicators and Reagents , Methylation , Molecular Sequence Data , Oligosaccharides/isolation & purification , TrypsinABSTRACT
A simple strategy was developed for the immunologic quantitative determination of small, biologically active peptides utilizing bradykinin (BK) as the model peptide prototype. Methods were developed for the preparation of a peptide-carrier complex suitable for immunization and for immobilization of peptides onto the plastic surface of enzyme-linked immunosorbent assay (ELISA) plates. An avidin-bound biotinylated peptide complex was used for raising peptide antibodies with high titers (1:4000) in the rabbit. The peptide BK was coupled to synthetic polymeric carriers poly-D-lysine (PL) and poly-D-lysine-succinylated (PLS) via the BK carboxy and amino terminus, respectively, with the aid of a water soluble carbodiimide. These carriers with antigen peptide side chains as well as avidin-biotinyl-peptide complexes were efficient surface immobilizing reagents for microwell plastic plates used in the detection of kinins by ELISA. Monoclonal antibodies reacted competitively with kinins in plates coated with either PL-BK or PLS-BK. In contrast, rabbit (polyclonal) antibodies reacted specifically in the plates coated with PLS-BK but only a non-specific reaction could be obtained with the PL-BK coated plates (i.e., could not be displaced with BK). Based on results using synthetic BK analogues, the carboxy terminal half of the BK molecule appears to be the stronger antigenic determinant in both mouse and rabbit systems. The polyclonal antibodies demonstrated a greater affinity to bradykinin compared to the monoclonal antibodies. Their use improved the sensitivity of the ELISA for kinin determination by one order of magnitude. Kinin levels determined in plasma tryptic digests by ELISA with the polyclonal antibodies and PLS-BK system were in agreement with published values.
Subject(s)
Bradykinin/analysis , Immunologic Techniques , Peptides/analysis , Amines , Animals , Antibodies, Monoclonal/immunology , Avidin/analogs & derivatives , Binding, Competitive/immunology , Biotin/analogs & derivatives , Bradykinin/analogs & derivatives , Bradykinin/immunology , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Humans , Kinins/analysis , Mice , Peptides/immunology , Polylysine/analogs & derivatives , Polylysine/chemistry , Polylysine/immunology , RabbitsABSTRACT
Two distinct alpha-(1-3)-fucosyltransferase activities have been identified in the colon carcinoma cell lines HT-29 and COLO-205. While both enzymatic activities exhibit similar affinities for a synthetic alpha-(1-3) acceptor and GDP-fucose, they differ with respect to divalent cation requirements, N-ethylmaleimide inhibition, and glycoprotein substrate specificity. The COLO-205 alpha-(1-3) activity exhibits maximal enzymatic activity in the presence of 20 mM Mn2+ but retains less than 10% activity in the absence of divalent cations. In contrast, the optimal Mn2+ concentration for the HT-29 enzyme is 1 mM, although this activity is relatively insensitive to divalent cation stimulation. In addition, the HT-29 alpha-(1-3)-fucosyltransferase activity is resistant to inhibition by 30 mM N-ethylmaleimide and relatively inactive toward the glycoprotein substrate fetuin as compared to its desialylated derivative, asialofetuin. The COLO-205 activity is inhibited approximately 90% by N-ethylmaleimide and is equally active with either glycoprotein acceptor. Although the alpha-(1-3) specific activities are similar in both cell lines, N-ethylmaleimide-sensitive alpha-(1-4) fucosyltransferase activity is 40-fold higher in COLO-205 as compared to HT-29, suggesting that the COLO-205 fucosyltransferase activity may be an alpha-(1-3/4) enzyme, while the HT-29 activity appears to be an alpha-(1-3) specific form. Further examination of a panel of cell lines, tumor biopsies, and xenografts, based on the effect of metal ions and N-ethylmaleimide, indicated that both enzyme activities are similarly expressed in human colon carcinoma tissue.