Kalyan R. Anumula

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.

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography

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.

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units☆

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H2(35)SO4. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the alpha and beta subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [35S]sulfate was also incorporated into several other proteins in addition to LH. The location of 35SO2-(4) in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[35S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[35S] sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of 35SO2-(4) by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.

Single tag for total carbohydrate analysis

Anthranilic acid (2-aminobenzoic acid, 2-AA) has the remarkable property of reacting rapidly with every type of reducing carbohydrate. Reactivity of 2-AA with carbohydrates in aqueous solutions surpasses all other tags reported to date. This unique capability is attributed to the strategically located -COOH which accelerates Schiff base formation. Monosaccharides, oligosaccharides (N-, O-, and lipid linked and glycans in secretory fluids), glycosaminoglycans, and polysaccharides can be easily labeled with 2-AA. With 2-AA, labeling is simple in aqueous solutions containing proteins, peptides, buffer salts, and other ingredients (e.g., PNGase F, glycosidase, and transferase reaction mixtures). In contrast, other tags require relatively pure glycans for labeling in anhydrous dimethyl sulfoxide-acetic acid medium. Acidic conditions are known to cause desialylation, thus requiring a great deal of attention to sample preparation. Simpler labeling is achieved with 2-AA within 30-60 min in mild acetate-borate buffered solution. 2-AA provides the highest sensitivity and resolution in chromatographic methods for carbohydrate analysis in a simple manner. Additionally, 2-AA is uniquely qualified for quantitative analysis by mass spectrometry in the negative mode. Analyses of 2-AA-labeled carbohydrates by electrophoresis and other techniques have been reported. Examples cited here demonstrate that 2-AA is the universal tag for total carbohydrate analysis.

Identification of attachment sites and structural classes of asparagine-linked carbohydrates in glycoproteins.

Publisher Summary This chapter discusses identification of attachment sites and structural classes of asparagine-linked carbohydrates in glycoproteins. Glycosylation of specific asparagine (Asn) residues is one of the most common posttranslational modifications of proteins. However, its importance is just now becoming understood and widely appreciated. Current theories concerning the roles of Asn-linked oligosaccharides in modulating the biological activity of glycoproteins have been recently reviewed. The attachment-site Asn residues are with few exceptions present in an Asn-X-Ser/Thr consensus sequence in which “X” may be any amino acid except proline. In contrast to the peptide backbone of the protein, structural heterogeneity of the Asn-linked oligosaccharides is the rule rather than the exception. Glycoproteins commonly have several different structural classes of oligosaccharide attached (i.e., oligomannose, hybrid, or complex). Furthermore, each glycosylation site often has a heterogenous population of oligosaccharides of the same structural class, but differing in the number and type of sugar residues attached to the ends of the chains or antennae. The chapter describes two interrelated mass spectrometry-based strategies for: (1) identifying the attachment sites of Asn-linked sugars in glycoproteins and extent of glycosylation at each site, and (2) defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site in glycoproteins.

Fluorescent N-methylanthranilyl (Mantyl) tag for peptides : its application in subpicomole determination of kinins

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.

[67] Keyhole limpet oligosaccharyl sulfatase

Publisher Summary This chapter focuses on obtaining a purified preparation of an oligosaccharyl sulfatase capable of specific hydrolysis of the sulfate esters in macromolecules because chemical methods of desulfation of oligosaccharides are quite drastic and cause denaturation of proteins. In the context of this chapter, 3 H-labeled oLH (ovine luteinizing hormone) reduced oligosaccharides and intact 35 SO 4 -1abeled oLH have been used as substrates for the purification of an oligosaccharyl sulfatase with the anticipation that such an enzyme will be of general application. From among the several commercially available crude or purified sulfatase preparations, it was found that the keyhole limpet acetone powder contained the desired enzyme. The chapter discusses describes the purification of such a sulfatase and some of its properties. The data in the chapter suggest that the limpet acetone poweder contains a specific enzyme that hydrolyzes sulfate ester in the asparagines-linked oligosaccharides preferentially to p-nitrocatechol sulfate. Keyhole limpet is a good source for the isolation of the sulfatase that would act on asparagine-linked carbohydrate structures containing sulfate esters. It may provide a useful tool in probing the functional role of sulfate ester in glycoproteins.