Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.
"Dimerization of viral proteins may be important for authentic antigen presentation to the host immune system and for induction of a stable, long-term immunity. Lectin analysis of the PK15 cells-derived E2his revealed a glycosylation pattern basically composed of hybrid and complex N-glycans with sialic acid ␣(2—3) and ␣(2—6) capping the non-reducing terminals, which is in agreement with the glycosylation patterns of pig kidney cells . Thus, the Nlinked glycosylation pattern of the PK15 cells-derived E2his probably resembles the glycosylation pattern present in the E2 produced during the infection in the CSFV natural host. "
[Show abstract][Hide abstract] ABSTRACT: E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.
[Show abstract][Hide abstract] ABSTRACT: This review describes the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates and continues coverage of the field from the previous review published in 1999 (D. J. Harvey, Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates, 1999, Mass Spectrom Rev, 18:349-451) for the period 1999-2000. As MALDI mass spectrometry is acquiring the status of a mature technique in this field, there has been a greater emphasis on applications rather than to method development as opposed to the previous review. The present review covers applications to plant-derived carbohydrates, N- and O-linked glycans from glycoproteins, glycated proteins, mucins, glycosaminoglycans, bacterial glycolipids, glycosphingolipids, glycoglycerolipids and related compounds, and glycosides. Applications of MALDI mass spectrometry to the study of enzymes acting on carbohydrates (glycosyltransferases and glycosidases) and to the synthesis of carbohydrates, are also covered.
Mass Spectrometry Reviews 07/2006; 25(4):595-662. DOI:10.1002/mas.20080 · 7.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A simple microadsorption technique is described to remove detergent additives from oligosaccharide samples before their mass spectrometric analysis. The described methodology has been validated with submicrogram quantities of contaminated glycoproteins. This procedure is applicable to investigating minute quantities of glycans in both the positive- and negative-ion mode of matrix-assisted laser desorption/ionization mass spectrometry.
Rapid Communications in Mass Spectrometry 07/2000; 14(14):1233-7. DOI:10.1002/1097-0231(20000730)14:14<1233::AID-RCM16>3.0.CO;2-P · 2.25 Impact Factor
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